Neuronal overexpression of DYRK1A/minibrain alters motor decline, neurodegeneration and synaptic plasticity in Drosophila

Down syndrome (DS) is characterised by abnormal cognitive and motor development, and later in life by progressive Alzheimer’s disease (AD)-like dementia, neuropathology, declining motor function and shorter life expectancy. It is caused by trisomy of chromosome 21 (Hsa21), but how individual Hsa21 genes contribute to various aspects of the disorder is incompletely understood. Previous work has demonstrated a role for triplication of the Hsa21 gene DYRK1A in cognitive and motor deficits, as well as in altered neurogenesis and neurofibrillary degeneration in the DS brain, but its contribution to other DS phenotypes is unclear. Here we demonstrate that overexpression of minibrain (mnb), the Drosophila ortholog of DYRK1A, in the Drosophila nervous system accelerated age-dependent decline in motor performance and shortened lifespan. Overexpression of mnb in the eye was neurotoxic and overexpression in ellipsoid body neurons in the brain caused age-dependent neurodegeneration. At the larval neuromuscular junction, an established model for mammalian central glutamatergic synapses, neuronal mnb overexpression enhanced spontaneous vesicular transmitter release. It also slowed recovery from short-term depression of evoked transmitter release induced by high-frequency nerve stimulation and increased the number of boutons in one of the two glutamatergic motor neurons innervating the muscle. These results provide further insight into the roles of DYRK1A triplication in abnormal aging and synaptic dysfunction in DS. Author summary Down syndrome (DS) is caused by three copies of chromosome 21 instead of the usual two. It is characterised by cognitive and motor deficits, which worsen with age resulting in Alzheimer’s disease (AD). Which genes on chromosome 21 cause these phenotypes is incompletely understood. Here we demonstrate that neuronal overexpression of minibrain, the Drosophila ortholog of the chromosome 21 gene DYRK1A, causes age-dependent degeneration of brain neurons, accelerates age-dependent decline in motor performance and shortens lifespan. It also modifies presynaptic structure, enhances spontaneous transmitter release and slows recovery from short-term depression of synaptic transmission at a model glutamatergic synapse. These findings give insight into the role of DYRK1A overexpression in aberrant aging and altered information processing in DS and AD.


Abstract
Down syndrome (DS) is characterised by abnormal cognitive and motor development, and later in life by progressive Alzheimer's disease (AD)-like dementia, neuropathology, declining motor function and shorter life expectancy. It is caused by trisomy of chromosome 21 (Hsa21), but how individual Hsa21 genes contribute to various aspects of the disorder is incompletely understood. Previous work has demonstrated a role for triplication of the Hsa21 gene DYRK1A in cognitive and motor deficits, as well as in altered neurogenesis and neurofibrillary degeneration in the DS brain, but its contribution to other DS phenotypes is unclear. Here we demonstrate that overexpression of minibrain (mnb), the Drosophila ortholog of DYRK1A, in the Drosophila nervous system accelerated age-dependent decline in motor performance and shortened lifespan. Overexpression of mnb in the eye was neurotoxic and overexpression in ellipsoid body neurons in the brain caused age-dependent neurodegeneration. At the larval neuromuscular junction, an established model for mammalian central glutamatergic synapses, neuronal mnb overexpression enhanced spontaneous vesicular transmitter release. It also slowed recovery from short-term depression of evoked transmitter release induced by high-frequency nerve stimulation and increased the number of boutons in one of the two glutamatergic motor neurons innervating the muscle. These results provide further insight into the roles of DYRK1A triplication in abnormal aging and synaptic dysfunction in DS.

Introduction
Down syndrome (DS, also known as Down's syndrome) or trisomy 21 is caused by the presence of three copies of chromosome 21 (Hsa21) instead of the usual two [1]. It is characterised by cognitive impairment [2] and the delayed and incomplete acquisition of motor skills [3] as a result of abnormal development of the nervous system [4]. Individuals with DS almost invariably develop Alzheimer's disease (AD)-like symptoms (AD-DS). These include progressive dementia after 40 years of age, the onset of amyloid plaques, neurofibrillary tangles and neurodegeneration after 10 -20 years [5,6], faster age-dependent motor decline that is an early marker for the onset of cognitive decline and health deterioration [7,8], and a shorter mean life expectancy by approximately 28 years [9]. Currently there is no treatment for DS or AD; our understanding of the mechanisms of the disorder is incomplete and this hampers the development of effective therapies.
One of the Hsa21 genes, DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A), is a candidate causative gene for the structural and functional changes that occur in the DS brain, and for the associated cognitive and motor deficits [1,4]. DYRK1A/Dyrk1a mRNA and protein are expressed throughout the brain in humans and rodents, wherein DYRK1A controls aspects of neuronal development and function [10][11][12]. DYRK1A/Dyrk1a mRNA and protein expression is increased in DS brain and in the brain of different mouse models of DS [10][11][12][13], whether the gene is triplicated as part of a genomic segment, as in Dp1Yey, Ts65Dn, Ts1Cje and Tc1 mice, or alone as in TgDyrk1a and TgDYRK1A mice [1].
However, the contribution of DYRK1A overexpression to the faster age-dependent decline in cognitive and motor function in DS is unclear. It is predicted to play a role as Dyrk1a overexpression intensifies with age in the brain of Ts65Dn and Ts1Cje mouse models of DS [12,15,23,24], it is associated with AD-DS-like histopathological changes in the brain of aged Ts65Dn mice [5,25] and it enhances APP processing and the formation of hyperphosphorylated Tau aggregates in rat hippocampal progenitor cells in vitro [26]. The contribution of DYRK1A overexpression to the shorter life expectancy in DS has also not been explored.
Cognitive and motor dysfunction in individuals with DS and in mouse models of DS are associated with changes in synaptic plasticity and with changes in the number and structure of GABAergic and glutamatergic brain neurons and synapses [10,27,28]. Such modifications have been linked to Dyrk1a overexpression [10,13,25,29], but the effects of Dyrk1a overexpression on the basic properties of synaptic function have rarely been explored. In one study, there was no change in the frequency of miniature excitatory synaptic currents (mEPSCs) or the probability of electrically-evoked glutamate release in the prefrontal cortex of TgDyrk1a mice [30]. Nevertheless, since Dyrk1a controls the activity of proteins that regulate endocytosis [31] and DYRK1A overexpression slows endocytosis of transmitter vesicles in hippocampal presynaptic membranes from TgDYRK1A mice [32], modulation of transmitter release at other glutamatergic synapses is likely.
To investigate the contribution of DYRK1A overexpression in the nervous system to various aspects of DS, we overexpressed minibrain (mnb), the Drosophila ortholog of DYRK1A [10], in the Drosophila nervous system and implemented well-established assays in larvae and adult flies [33][34][35]. The assays monitored motor impairment and its development with age, lifespan, age-related neurodegeneration, and synaptic dysfunction. Due to their short lifecycle, Drosophila are one of the pre-eminent models for aging and neurodegeneration [36], both aspects of DS that are more difficult to investigate in mice.
The Drosophila larval neuromuscular junction (NMJ) is a well-established model for mammalian central glutamatergic synapses and is easily accessible to electrophysiology [33]. Mnb is expressed presynaptically at larval NMJs and reducing its expression changes motor nerve terminal structure and impairs recycling of transmitter vesicles [37]. Overexpression of the mnb-F transcript induces a different change in nerve terminal morphology without apparently affecting basal synaptic transmission [37]. Here we report the effects of neuronal overexpression of mnb-H, which encodes the longest mnb splice variant [38], on motor function, the rate of motor decline with age, lifespan, age-related neurodegeneration, presynaptic structure, spontaneous transmitter release and recovery from frequency-dependent depression of electrically-evoked transmitter release.

Neuronal overexpression of mnb produced motor deficits in larvae, accelerated age-dependent motor decline in adult flies and shortened adult lifespan
The effect of mnb overexpression in the nervous system on motor function (specifically the mnb-H splice variant) was tested using two assays of fly larval locomotion. Elav>mnb larvae, overexpressing mnb throughout the nervous system under the control of the Elav-Gal4 driver [39], did not move as far as control larvae (Elav/+) in a free movement assay (Fig. 1A), which measures the ability of larvae to perform rhythmic muscle contractions necessary for gross locomotion [40]. They also took longer to complete a self-righting assay (Fig. 1B), which is a more complex motor task requiring larvae to enact a co-ordinated sequence of movements to right themselves after being rolled onto their backs [41]. To assess the impact of neuronal mnb overexpression on age-related decline in locomotor function, the performance of the same cohorts of adult flies was assessed in a negative geotaxis assay at different ages [36]. This showed acceleration in Elav>mnb flies of the usual age-related decline in performance (Elav/+). There was also evident shortening of the lifespan of Elav>mnb flies, so that the median lifespan was reduced by almost 50% (Elav/+, 73 days; Elav>mnb, 38 days; Fig. 1D). These results indicate that neuronal overexpression of mnb alone produced a motor deficit and abnormal aging characterised by accelerated age-related locomotor impairment and a shorter lifespan.

Overexpression of mnb caused neurodegeneration in adult flies
As DYRK1A triplication has been linked to degeneration of brain neurons in AD-DS and in Ts65Dn mice [25,42], we tested the possibility that neuronal overexpression of mnb is sufficient to cause neurotoxicity and age-related neurodegeneration using two established assays of neurodegeneration in adult flies [34,35]. In the first, mnb was overexpressed in the eye through development and adulthood using the Glass multimer reporter driver (GMR-Gal4) [43]. The GMR>mnb flies, but not control flies (GMR/+), had a reduced eye surface area and a visible "rough eye" phenotype ( Fig. 2A), both of which indicate neural death and the resultant breakdown of the regularly spaced array of ommatidia making up the retina. In a second assay, the EB1 driver (EB1-Gal4) was used to overexpress mnb in the ellipsoid body (EB), a subpopulation of neurons within the central complex of the brain implicated in locomotor control (Fig. 2B) [44]. The EB cells also expressed membrane-bound GFP which enabled their visualisation. At 1 day old, there was no difference in the number of GFP-positive EB neurons between control (EB1/+) and EB1>mnb flies, whereas at day 40 the number of EB neurons was significantly reduced in EB1>mnb flies but not in control flies, and the remaining cells were fragmented and misshapen (Fig. 2C). Therefore, neurotoxicity caused by mnb overexpression promoted age-related neurodegeneration in a central neuron population.

Overexpression of mnb in motor neurons increased the number of synaptic boutons at the larval NMJ
To investigate the effect of mnb overexpression on presynaptic morphology, mnb was overexpressed in glutamatergic motor neurons of Drosophila larvae using OK371-Gal4 [45]. The neuronal membranes were labelled with horseradish peroxidase (HRP) and boutons of the two motor neurons innervating larval muscles 6 and 7 were differentiated according to stronger postsynaptic expression of Discs large (Dlg) opposite 1b (big) boutons than 1s (small) boutons [46]. Analysis of the NMJ in the second abdominal larval segment, A2, showed that mnb overexpression affected the morphology of the nerve terminals of only one of the motor neurons; it increased the number of 1b boutons but did not alter the number of 1s boutons ( Fig. 3A-B). The effect was not secondary to changes in muscle size, as this did not differ (surface area of muscle 6: OK371/+, 44752 ± 1407 μm 2 , n = 15; OK371>mnb, 44681 ± 3684 μm 2 , n = 15, P = 0.9857).

Overexpression of mnb in motor neurons altered basal synaptic transmission at the larval NMJ
As neuronal overexpression of mnb increased the number of 1b boutons at the larval NMJ, and because previous studies have implicated Dyrk1a/mnb in the control of the recycling of neurotransmitter vesicles [31,32,37], we investigated if spontaneous glutamate release was altered by recording spontaneously occurring miniature excitatory junction potentials (mEJPs) with intracellular microelectrodes. Since the muscle 6/7 NMJ is innervated by 1b and 1s boutons, mEJPs usually result from the release of neurotransmitter vesicles from both types of boutons [47]. Our recordings revealed that mEJPs occurred more frequently but were smaller in OK371>mnb larvae ( Fig.   4A-B). The decrease in amplitude was not secondary to a change in the electrical properties of the muscle as there was no difference in input resistance (OK371/+, 3.37 ± 0.69 MΩ, n = 8; OK371>mnb = 3.99 ± 0.99 MΩ, n = 8, P = 0.613) or resting potential (OK371/+, -69.6 ± 1.48 mV, n = 8; OK371>mnb, -67.6 ± 1.55 mV, n = 8, P = 0.365). Although we did not directly investigate the source of the more frequent smaller mEJPs, the notion that they are due to the observed selective increase in the number of 1b boutons is suggested by the fact that 1b-dependent mEJPs are smaller than 1s-dependent mEJPS [47]. In parallel with the changes in spontaneous synaptic events, there was a small (~11 %) decrease in the mean amplitude of electrically-evoked excitatory junction potentials (EJPs) caused by single stimuli applied to the nerve at a low frequency (0.1 Hz) (Fig. 4C). There was no difference between EJPs in mean rise time (OK371/+, 2.67 ± 0.178 ms, n = 8; OK371>mnb, 3.23 ± 0.33 ms, n = 8, P = 0.728) or mean time constant of decay (OK371/+, 44.9 ± 3.1 ms, n = 8; OK371>mnb, 36.6 ± 4.6 ms, n = 8, P = 0.154). The relatively small fall in EJP amplitude is likely to reflect the smaller size of the 1b-dependent component of the EJP relative to that of the 1s-dependent component [47].

Overexpression of mnb in motor neurons slowed recovery from frequency-dependent depression at the larval NMJ
To investigate the effects of neuronal mnb overexpression on recycling of synaptic vesicles during electrically-evoked transmitter release, EJPs were evoked with pairs of electrical stimuli separated by intervals of varying duration (10 ms -10 s) or with repeated trains of 10 stimuli applied at a high frequency (10 Hz, a frequency 100 times higher than that at which the single EJPs were evoked) [48].
At control NMJs, paired pulses separated by intervals shorter than 200 ms caused depression of the amplitude of the second EJP relative to that of the first and the depression was stronger for shorter inter-stimulus intervals (Fig. 5A). The dependence of paired-pulse depression on interval duration was unaltered in OK371>mnb larvae (Fig. 5A), indicating that mnb overexpression did not alter release from a readily releasable pool of vesicles [49]. When transmitter release was evoked at control NMJs with a train of 10 stimuli at 10 Hz, there was rapid depression of the EJP amplitude by ~20% within the first 3 events (Fig. 5B). In the one-minute interval before the next train, the EJP amplitude recovered fully so that the amplitude of the first EJP in the second train was the same as in the first train (Fig.   5B). This ability to recover did not wane during the recording; the amplitude of the first EJP in each train did not differ between 8 trains (Fig. 5B). These effects are consistent with previous studies [48] and confirm rapid depletion and replenishment of the readily releasable pool of vesicles [49].
However, the same pattern of nerve stimulation produced different effects at OK371>mnb NMJs (Fig.   5B). The percentage decrease in EJP amplitude during each train was the same as at control NMJs, but the depression was not fully reversed during the intervals between trains, so that the first EJP in each train was smaller than the first EJP in the preceding train. The depression in amplitude accumulated over the 8 trains, resulting in an overall fall of 10%. To confirm that the changes in EJP amplitude were due to presynaptic changes in transmitter release and were not postsynaptically mediated by a decrease in the unitary depolarisations comprising each EJP, we measured the amplitudes of 200 mEJPs immediately before and 200 mEJPs immediately after the series of trains at each NMJ. At both control and OK371>mnb NMJs, the cumulative distribution of mEJP amplitudes before and after a series of trains was similar; although they were not identical, the observed slight increase in the number of larger mEJPs cannot explain the decline in EJP amplitude (Fig. 5C). These results show that mnb overexpression slowed replenishment of the readily releasable pool of vesicles, an effect consistent with the reported slowing of endocytosis of transmitter vesicles by DYRK1A overexpression [32].

Discussion
This study demonstrated that neuronal overexpression of mnb, the Drosophila ortholog of DYRK1A, is sufficient to induce motor impairment, accelerate age-related decline in motor performance, shorten lifespan and cause age-dependent neurodegeneration. This study also found that neuronal mnb overexpression at a glutamatergic synapse alters presynaptic structure, modifies basal synaptic transmission and delays recovery from short-term synaptic depression.
People with DS have impaired motor skills which are evident from childhood and are caused by abnormal development of the nervous system [3,4]. Later, in middle age, they undergo faster agedependent motor decline, which is an early marker of future dementia, comorbidities and mortality, and is likely caused by histopathological changes in the brain [7,8]. The life expectancy of people with DS is about 28 years shorter than the general population [9]. By taking advantage of the relatively short life cycle of Drosophila and driving overexpression of mnb in neurons, we have demonstrated a potential role for neuronal DYRK1A overexpression in the accelerated age-dependent decline of motor function and shortening of life expectancy in DS. The genetic basis of these aspects of DS is more difficult and costly to explore in mouse models of DS, due to the longer time required to study aged mice. Our finding that mnb overexpression causes age-related neurodegeneration confirms previous studies inferring a link between DYRK1A overexpression and degeneration and loss of neurons [10,15,25,42], which is associated with faster age-related decline in motor and cognitive function in DS and AD-DS. Our results also reinforce the conclusion from earlier studies with adult mice overexpressing DYRK1A or Dyrk1a, alone or as part of a chromosomal segment, that triplication of DYRK1A is likely to contribute to motor deficits in DS [13][14][15][16][17][18][19].
In addition to the smaller brain size and fewer brain neurons in DS and mouse models of DS, there are alterations in the structure of brain synapses that are predicted to modify synaptic function [4,28,50]. A previous study showed that DYRK1A overexpression in mice changes postsynaptic morphology in the cortex and in cultured cortical neurons by reducing the number and length of dendrites and by reducing the number of dendritic spines but elongating their shape [51]. It also decreased the number of synapses formed. Our study shows that mnb overexpression changes presynaptic structure and that this happens in a neuron-specific manner; mnb overexpression in the two glutamatergic motoneurons innervating the larval NMJ increased the number of 1b boutons without changing the number of 1s boutons. These data are consistent with a previous study which demonstrated that reduced levels of mnb caused a decrease, and increased levels of the mnb-F transcript an increase, in the number of boutons at the NMJ [37], but did not differentiate between 1b and 1s boutons.
The cognitive and motor deficits in DS arise from aberrant information processing in the brain that is likely due, in part, to changes in synaptic transmission or synaptic plasticity. Individuals with DS have impaired synaptic plasticity in the motor cortex [27]. Our finding that mnb overexpression slows replenishment of the readily releasable pool of vesicles, and also modifies basal synaptic transmission, confirms a previous suggestion that DYRK1A overexpression contributes to synaptic dysfunction and cognitive deficits associated with DS, made on the basis of the observed slowing of endocytosis of transmitter vesicles in cultured mouse hippocampal neurons overexpressing human DYRK1A [32]. The effects of DYRK1A on synaptic function may be splice variant specific as we found that overexpression of the mnb-H transcript caused a decrease in mEJP and EJP amplitude, whereas overexpression of mnb-F in a previous study caused an increase in mEJP amplitude and no change in EJP amplitude at the larval NMJ [37].
The effects of neuronal mnb overexpression on larval NMJ function replicate some, but not all, the documented changes in glutamatergic synaptic transmission in the brain of mouse models of DS.
These include a decrease in the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in neocortical neurons of Ts65Dn mice [52], compromised glutamate release in response to stimuli trains at hippocampal CA1 synapses of Ts1Cje mice [53] and a decrease in EPSC amplitude in hippocampal CA3 neurons of Ts65Dn mice [54]. However, in contrast to the increase in mEJP frequency caused by mnb overexpression at the larval NMJ, electrophysiological studies have found a decrease in the frequency of mEPSCs in hippocampal CA3 neurons of Ts65Dn mice, sEPSCs in neocortical neurons of Ts65Dn mice and sEPSCs in neurons derived from trisomy 21 induced pluripotent stem cells, or no change in mEPSC frequency in the prefrontal cortex of TgDyrk1a mice or mossy fibre-CA3 synapses in Tc1 mice [28].
Our study further elucidates the role of DYRK1A triplication in various DS phenotypes. It supports the future development of pharmacological inhibitors of DYRK1A as treatments for multiple aspects of DS and AD [10,12]. Further work is necessary to fully understand interactions between DYRK1A and other triplicated Hsa21 genes in DS, in specific cell types and during defined periods of development and ageing. Behaviour mnb expression was driven throughout the nervous system using Elav-Gal4 [39] for experiments investigating behaviour of wandering third instar larvae, the number of boutons at the larval NMJ, and synaptic transmission at the larval NMJ. All behavioural experiments took place at 25 o C. Larval locomotor experiments were conducted on a 9.5 cm petri dish containing 1.6% agarose. A single third instar wandering larva was selected, washed in a drop of distilled H 2 O, transferred to the agarose and allowed 30 s to acclimatise. To analyse free movement, the dish was placed over a 0.5 cm grid and the number of lines the larva crawled across in one minute was counted by eye. The self-righting assay was conducted as described elsewhere [41,55]; the larva was gently rolled onto its back on the agarose using a fine moistened paintbrush, held for one second and released, and the time for it to right itself was recorded.

Animals
The negative geotaxis assay was performed as described previously [56]. A cohort of 10 flies was transferred without anaesthesia to an empty 9.5 cm tube with a line drawn 2 cm from the top. After 1 minute acclimatisation, the vial was sharply tapped 3 times to knock the flies to bottom. The number of flies to climb past the line within 10 s was recorded. 15 cohorts of 10 flies were tested for each genotype. Age-dependent changes in climbing were assessed by repeating the negative geotaxis assay at 10, 20 and 30 days post-eclosure [57]. For the survival assay, 10 cohorts of 10 once-mated females were transferred to a vial of fresh food twice weekly and the number of surviving flies recorded at each transfer.

Antibody staining and visualisation at the NMJ
Wandering third instar larvae were dissected in ice-cold, Ca 2+ -free HL3.1-like solution (in mM: 70 NaCl, 5 KCl, 10 NaHCO 3 , 115 sucrose, 5 trehalose, 5 HEPES, 10 MgCl 2 ) to produce a larval "fillet" [58]. The fillet was fixed for 30 min in 4% paraformaldehyde (Sigma Labs), washed three times in 1% Triton-X (Sigma Labs) and blocked for one hour in 5% normal goat serum (Fitzgerald Industries) and 1% Triton-X at room temperature. It was incubated overnight in 1/500 FITC-conjugated anti-horseradish peroxidase (HRP-FITC) (Jackson Immunoresearch Laboratories) and 1/500 mouse anti Discs large (Dlg) primary antibody, then for two hours in 1/500 AlexaFluor 633-conjugated goat anti-mouse secondary antibody at room temperature. Each fillet was washed and mounted on a coverslip in Vectashield (Vector Laboratories). Z-series of NMJs were imaged on a Leica SP5-II confocal laser-scanning microscope using an oil immersion 40 × objective. The number of boutons at the NMJ of muscle 6/7 in segment A2 was counted manually. ImageJ (rsb.info.nih.gov/ij/) was used to manually outline muscle 6 and hence calculate their area.

Neurotoxicity
Overexpression of mnb was driven in the eye using the Glass multimer reporter (GMR-Gal4). Images of the whole head of 1-2 day old flies were taken via a Zeiss AxioCam MRm camera attached to a stereomicroscope (Zeiss SteREO Discovery.V8, up to 8× magnification), and the surface area of the eye was calculated by manually outlining the eye in ImageJ (rsb.info.nih.gov/ij/). Overexpression of mnb in GFP-tagged ellipsoid body (EB) ring neurons was achieved by crossing EB1-Gal4; UAS-mCD8-GFP flies with UAS-mnb flies. Following published methods [59], adult brains were dissected, fixed for 30 minutes in 4% paraformaldehyde and mounted on a coverslip in Vectashield (Vector Laboratories).
Slides were imaged on a Leica SP5-II confocal laser scanning microscope using an oil immersion 40 × objective. A Z-stack of 25 images at 1 μm increments was captured and combined into a 3-D projection using ImageJ (rsb.info.nih.gov/ij/); analysis was performed by scrolling through all 25 images and counting the number of intact cells in one brain hemisphere.

Electrophysiology
Wandering third instar larvae were dissected as for antibody staining. The motor nerves were severed just below the ventral ganglion and the brain was removed.

Statistical analysis
Statistical analysis was conducted in GraphPad Prism (v. 6