Learning Spectral Fractional Anisotropy and Mean Diffusivity Features as Neuroimaging Biomarkers for Tracking White Matter Integrity Changes in Myotonic Dystrophy Type 1 Patients using Deep Convolutional Neural Networks.

Myotonic dystrophy type 1 (DM1) is a genetic neuromuscular progressive multisystem disease that results in a broad spectrum of clinical central nervous system (CNS) involvement, including problems with memory, attention, executive functioning, and social cognition. Fractional anisotropy and mean diffusivity along-tract data calculated using diffusion tensor imaging techniques play a vital role in assessing white matter microstructural changes associated with neurodegeneration caused by DM1. In this work, a novel spectrogram-based deep learning method is proposed to characterize white matter network alterations in DM1 with the goal of building a deep learning model as neuroimaging biomarkers of DM1. The proposed method is evaluated on fractional anisotropies and mean diffusivities along-tract data calculated for 25 major white matter tracts of 46 DM1 patients and 96 unaffected controls. The evaluation data consists of a total of 7100 spectrogram images. The model achieved 91% accuracy in identifying DM1, a significant improvement compared to previous methods.Clinical relevance- Clinical care of DM1 is particularly challenging due to DM1 multisystem involvement and the disease variability. Patients with DM1 often experience neurological and psychological symptoms, such as excessive sleepiness and apathy, that greatly impact their quality of life. Some of DM1 CNS symptoms may be responsive to treatment. The goal of this research is to gain a deeper understanding of the impact of DM1 on the CNS and to develop a deep learning model that can serve as a biomarker for the disease, with the potential to be used in future clinical trials as an outcome measure.

A constant light-genetic screen identifies KISMET as a regulator of circadian photoresponses.

Circadian pacemakers are essential to synchronize animal physiology and behavior with the dayrationight cycle. They are self-sustained, but the phase of their oscillations is determined by environmental cues, particularly light intensity and temperature cycles. In Drosophila, light is primarily detected by a dedicated blue-light photoreceptor: CRYPTOCHROME (CRY). Upon light activation, CRY binds to the pacemaker protein TIMELESS (TIM) and triggers its proteasomal degradation, thus resetting the circadian pacemaker. To understand further the CRY input pathway, we conducted a misexpression screen under constant light based on the observation that flies with a disruption in the CRY input pathway remain robustly rhythmic instead of becoming behaviorally arrhythmic. We report the identification of more than 20 potential regulators of CRY-dependent light responses. We demonstrate that one of them, the chromatin-remodeling enzyme KISMET (KIS), is necessary for normal circadian photoresponses, but does not affect the circadian pacemaker. KIS genetically interacts with CRY and functions in PDF-negative circadian neurons, which play an important role in circadian light responses. It also affects daily CRY-dependent TIM oscillations in a peripheral tissue: the eyes. We therefore conclude that KIS is a key transcriptional regulator of genes that function in the CRY signaling cascade, and thus it plays an important role in the synchronization of circadian rhythms with the dayrationight cycle.

A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila.

A fundamental property of circadian rhythms is their ability to persist under constant conditions. In Drosophila, the ventral Lateral Neurons (LNvs) are the pacemaker neurons driving circadian behavior under constant darkness. Wild-type flies are arrhythmic under constant illumination, but flies defective for the circadian photoreceptor CRY remain rhythmic. We found that flies overexpressing the pacemaker gene per or the morgue gene are also behaviorally rhythmic under constant light. Unexpectedly, the LNvs do not drive these rhythms: they are molecularly arrhythmic, and PDF--the neuropeptide they secrete to synchronize behavioral rhythms under constant darkness--is dispensable for rhythmicity in constant light. Molecular circadian rhythms are only found in a group of Dorsal Neurons: the DN1s. Thus, a subset of Dorsal Neurons shares with the LNvs the ability to function as pacemakers for circadian behavior, and its importance is promoted by light.

PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila.

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cry(b). The period mutant per(L) manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of per(L) flies, because a per(L); cry(b) strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.

USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.

Interactions between circadian neurons control temperature synchronization of Drosophila behavior.

Most animals rely on circadian clocks to synchronize their physiology and behavior with the day/night cycle. Light and temperature are the major physical variables that can synchronize circadian rhythms. Although the effects of light on circadian behavior have been studied in detail in Drosophila, the neuronal mechanisms underlying temperature synchronization of circadian behavior have received less attention. Here, we show that temperature cycles synchronize and durably affect circadian behavior in Drosophila in the absence of light input. This synchronization depends on the well characterized and functionally coupled circadian neurons controlling the morning and evening activity under light/dark cycles: the M cells and E cells. However, circadian neurons distinct from the M and E cells are implicated in the control of rhythmic behavior specifically under temperature cycles. These additional neurons play a dual role: they promote evening activity and negatively regulate E cell function in the middle of the day. We also demonstrate that, although temperature synchronizes circadian behavior more slowly than light, this synchronization is considerably accelerated when the M cell oscillator is absent or genetically altered. Thus, whereas the E cells show great responsiveness to temperature input, the M cells and their robust self-sustained pacemaker act as a resistance to behavioral synchronization by temperature cycles. In conclusion, the behavioral responses to temperature input are determined by both the individual properties of specific groups of circadian neurons and their organization in a neural network.

Ectopic CRYPTOCHROME renders TIM light sensitive in the Drosophila ovary.

The period (per) and timeless (tim) genes play a central role in the Drosophila circadian clock mechanism. PERIOD (PER) and TIMELESS (TIM) proteins periodically accumulate in the nuclei of pace-making cells in the fly brain and many cells in peripheral organs. In contrast, TIM and PER in the ovarian follicle cells remain cytoplasmic and do not show daily oscillations in their levels. Moreover, TIM is not light sensitive in the ovary, while it is highly sensitive to this input in circadian tissues. The mechanism underlying this intriguing difference is addressed here. It is demonstrated that the circadian photoreceptor CRYPTOCHROME (CRY) is not expressed in ovarian tissues. Remarkably, ectopic cry expression in the ovary is sufficient to cause degradation of TIM after exposure to light. In addition, PER levels are reduced in response to light when CRY is present, as observed in circadian cells. Hence, CRY is the key component of the light input pathway missing in the ovary. However, the factors regulating PER and TIM levels downstream of light/cry action appear to be present in this non-circadian organ.

From light to genes: moving the hands of the circadian clock.

Mammalian circadian rhythms are generated by the hypothalamic suprachiasmatic nuclei and finely tuned to environmental periodicities by neurochemical responses to the light-dark cycle. Light reaches the clock through a direct retinohypothalamic tract, primarily through glutamatergic innervation, and its action is probably regulated by a variety of other neurotransmitters. A key second messenger in circadian photic entrainment is calcium, mobilized through membrane channels or intracellular reservoirs, which triggers the activation of several enzymes, including a calcium/calmodulin-dependent protein kinase and nitric oxide synthase. Other enzymes activated by light are mitogen-activated- and cGMP-dependent protein kinase; all of the above have been reported to be involved in the circadian responses to nocturnal light pulses. These mechanisms lead to expression of specific clock genes which eventually set the phase of the clock and of clock-controlled circadian rhythms.

Diurnal, circadian and photic regulation of calcium/calmodulin-dependent kinase II and neuronal nitric oxide synthase in the hamster suprachiasmatic nuclei.

Mammalian circadian rhythms are entrained by light pulses that induce phosphorylation events in the suprachiasmatic nuclei (SCN). Ca(2+)-dependent enzymes are known to be involved in circadian phase shifting. In this paper, we show that calcium/calmodulin-dependent kinase II (CaMKII) is rhythmically phosphorylated in the SCN both under entrained and free-running (constant dark) conditions while neuronal nitric oxide synthase (nNOS) is rhythmically phosphorylated in the SCN only under entrained conditions. Both p-CaMKII and p-NOS (specifically phosphorylated by CaMKII) levels peak during the day or subjective day. Light pulses administered during the subjective night, but not during the day, induced rapid phosphorylation of both enzymes. Moreover, we found an inhibitory effect of KN-62 and KN-93, both CaMKII inhibitors, on light-induced nNOS activity and nNOS phosphorylation respectively, suggesting a direct pathway between both enzymes which is at least partially responsible of photic circadian entrainment.