Downregulation of CDK5 Signaling In the Dorsal Striatum Alters Striatal Microcircuits and Perturbs Circadian Behavior in Mice

Dysfunction of striatal dopaminergic circuits has been implicated in motor impairment as well as in Parkinson’s disease (PD)-related circadian perturbations that may represent an early prodromal marker of PD. Cyclin-dependent kinase 5 (CDK5) acts negatively on dopamine (DA) signaling in the striatum, suggesting a critical role in circadian and sleep disorders. Here, we used CRISPR/Cas9 gene editing to produce dorsal striatum (DS)-specic knockdown (KD) of the Cdk5 gene in mice (referred to as DS-CDK5-KD mice) to investigate its role in vivo. DS-CDK5-KD mice exhibited decits in locomotor activity and disturbances in daily rest/activity cycles. Additionally, Golgi staining of neurons in the DS revealed that Cdk5 deletion caused a reduction in dendrite length and functional synapses, which was conrmed by signicant downregulation of MAP2, PSD95 and synapsin I. Correlated with this, DS-CDK5-KD mice displayed reduced phosphorylation of Tau at Thr181. Furthermore, whole-cell patch-clamp recordings of green uorescent protein (GFP)-tagged neurons in the striatum of DS-CDK5-KD mice revealed a decrease in the frequency of spontaneous inhibitory post-synaptic currents and an alteration of the excitatory/inhibitory synaptic balance. Notably, anterograde labeling showed that CDK5 knockdown in the DS disrupted long-range projections to the secondary motor cortex, dorsal and ventral thalamic nuclei, and the basolateral amygdala, which are involved in the regulation of motor and circadian rhythms in the brain. These ndings support a critical role of CDK5 in the DS in maintaining the striatal neural circuitry underlying motor and circadian rhythms related to PD.


Introduction
Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized pathologically by the loss of dopaminergic neurons in the substantia nigra (SN) pars compacta [1]. The striatum is the main recipient of dopaminergic innervation from the SN and, accordingly, the impact of its loss on striatal microcircuitry has been extensively studied [2,3]. Dysfunction of striatal dopaminergic neural circuits has been implicated in the pathophysiology of both motor and non-motor symptoms in PD [4,5]. In addition to the classical motor symptoms, disturbances in daily rest/activity cycles are common non-motor symptoms in PD and may have a substantial impact on the quality of life [6,7]. It is estimated that rest/activity cycle disturbance in PD emerges when approximately 60% of nigral neurons have been lost and dopaminergic striatal content is reduced by 80% [7]. Therefore, restoration of the circadian program in PD has received the attention of the medical and research community [8].
Traditionally, dopamine has been associated with wake-promoting activity. Amphetamines promote wakefulness by enhancing dopamine (DA) release and preventing its reuptake by DA transporters, which further illustrates the wake-promoting effects of DA [9]. Moda nil as a wakefulness inducer has also been linked to dopaminergic activity [10,11]. The striatum is the main recipient of dopaminergic innervation from the SN and is composed primarily of GABAergic medium spiny projection neurons (MSNs) that express DA receptors, D 1 and D 2 [12,13]. Dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) is a highly enriched cytosolic protein in MSNs and is considered an important integrator of striatal cellular excitability and synaptic transmission [14]. Dopamine exerts bidirectional control on the phosphorylation state of DARPP-32 at Thr34; D 1 receptors stimulate and D 2 receptors inhibit this phosphorylation.
Additionally, cyclin-dependent kinase 5 (CDK5) phosphorylates DARPP-32 at another site, Thr75, which leads to dephosphorylation of DARPP-32 at Thr34 [15,16]. Therefore, CDK5 negatively regulates dopamine signaling in the striatum, indicating a functional role of CDK5 in regulating the rest/activity cycle. In addition, CDK5 is involved in the control of dendritic spine formation and cortical neurotransmission. For example, Cdk5 phosphorylates the NMDA receptor subunit, NR2B, to modulate synaptic transmission [17], and phosphorylates postsynaptic density protein 95 (PSD-95) to promote synaptic PSD-95 clustering and glutamate transmission [18]. These ndings indicate that aberrant hypoactivation of CDK5 may contribute to neural circuitry disorders in the human and rodent brain. In mouse models of PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment results in higher levels of CDK5 and its speci c activator, the P25 complex, in dopaminergic neurons, leading to neuronal death [19]. Furthermore, MPTP is toxic to dopaminergic neurons in the substantia nigra, causing irreversible PD symptoms, in which changes to striatal DA levels are major determinants of daily rest/activity cycle perturbations [20][21][22]. Thus, we predict that high levels of CDK5 will facilitate both DA involvement in the maintenance of wakefulness and inhibition of locomotion in PD, although the exact mechanisms underlying these effects are not completely understood.
Here, we aimed to assess the role of CDK5 in the regulation of circadian rest/activity rhythms using CRISPR/Cas9 gene editing to generate dorsal striatum (DS)-speci c Cdk5-de cient mice (referred to as DS-CDK5-KD mice). The DS was chosen because it is a major area of DA involvement in the early stages of motor processing, and DS disturbance may produce the well documented behavioral changes associated with PD [23]. In addition to assessing motor activity, we examined changes in circadian rhythms of sleep-wake behaviors in DS-CDK5-KD mice. Electrophysiological and anterograde labeling studies were used to examine whether CDK5 knockdown impairs cellular excitability and synaptic transmission in the striatal network, which appears to play a key role in the pathophysiology of sleep dysfunction in mice. We aimed to clarify the role of CDK5 in regulating sleep and circadian disturbances associated with PD.

Animals
Eight-week-old male C57BL/6 mice, initially weighing 18-20 g (Vital River Laboratories, Beijing, China), were group-housed in a controlled environment at 18-22°C and 40-60% humidity, with a 12:12-h light/dark cycle and access to food and water ad libitum. Enrichment was provided with shredding nestlets. Cages (32 × 22 × 17 cm, eight mice per cage) were changed every week by designated facility staff. All mice were group-housed for 1 week prior to use in an experiment and handled daily to minimize the effects of handling stress. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) and were approved by the Animal Care and Use Committees of the Beijing Institute of Pharmacology and Toxicology. Efforts were made to minimize the number of animals used for each experiment.

Experimental design and behavior test
Mice were screened 3 days before experiment, and mice with abnormal behavior were excluded. Mice were randomly divided into WT, DS-CDK5-NC and DS-CDK5-KD groups, and each group included 24 mice.
After nished lentivirus transduction at 14 th day, 8 mice from each group were selected for behavioral testing and 4 mice from each group were selected for immunoassay (Fig. 1C). The remaining mice were used for Golgi staining, electrophysiology assays or anterograde labeling. All behavioral experiments were performed at xed times (9:00-12:00 Am). Each behavioral testing was performed with an interval of 24 h in the same animals. The testing apparatus was cleaned with a hypochlorous acid solution between subjects. The experimenters were blinded to grouping and drug treatment.

Locomotor activity measurements
A locomotor activity test was used to assess spontaneous locomotor activity and arousal in mice. The method used was similar to a previously published protocol [25]. Brie y, animals were placed individually in a square arena (40 × 40 × 80 cm) with a Plexiglas oor and walls (Kinder Inc., Poway, CA, USA) and were allowed to move freely. After a 5 min habituation period, all animal locomotor activities were recorded with AnyMaze software (Stoelting Inc., USA), and distance traveled during a 30 min period was recorded and analyzed.

Wheel-running behavioral test
Mice were housed in light and temperature-controlled circadian cabinets (standard mouse circadian cabinet, Actimetrics, Wilmette, IL) within polypropylene cages (33.2 × 15 × 13 cm) containing a metal running wheel (11 cm diameter). Mice were acclimatized to the running wheel for 1 h prior to assessment. Locomotor activity rhythms were monitored with a ClockLab data collection system (Version 3.603, Actimetrics, Wilmette, IL) through the number of electrical closures triggered by wheel rotations. Cage changes were scheduled at 24-h intervals. Wheel-running activity was collected over a period of 24 h and analyzed using ClockLab Analysis software (Actimetrics Software).

Golgi staining and dendritic spine measurement
Golgi-Cox staining was performed using a Rapid Golgistain Kit (FD Neuro Technologies, Ellicott City, MD, USA) following the manufacturer's instructions. Brie y, brains were quickly removed and rinsed, and incubated in a mix of solution A/B for 14 days in the dark at room temperature. Then, solution A/B was changed to solution C for 3 days. Coronal sections of the DS (200 μm thick) were cut (ranging from 0.7-1.2 mm anterior to bregma; two sections per animal) on a freezing microtome (Leica, Wetzlar, Germany) and mounted onto gelatinized slides. After sections were dried in the dark, they were reacted in solutions D and E for 10 minutes, and dehydrated sequentially in 50, 75, 95 and 100% ethanol. Finally, sections were cleared in xylene and cover slipped with resinous mounting medium.
For dendritic spine measurement, the DS region was identi ed at low power (100 × magni cation), and MSNs were traced at 250 × ( nal magni cation) using the camera lucida technique on an Olympus light microscope (Model BX51) equipped with a drawing attachment. MSNs were traced using the 8-bit ImageJ plugin, Neuron J. Dendrite length and branching were measured using a Sholl analysis of ring intersections. A series of concentric rings at 20 µm increments printed on a transparency was centered over the soma. The total number of intersections between each ring and dendritic branches was counted and converted to estimates of dendrite length as a function of distance from the soma (i.e., for each 20 µm segment) and overall dendrite length. Spine density was measured manually in the stacks using the ImageJ Plugin, Cell Counter. Three to ve dendrite segments per slice, with each dendrite segment ranging from 20 to 30 μm in length, were used for spine density analysis. Spines were marked in the appropriate focal plane, preventing any double counting of spines.

Immunohistochemistry
After the last behavioral test, mice were euthanized and striatum sections were prepared for immunohistochemistry as previously described (Huang et al., 2016). Brie y, striatal sections (30 μm) were cut with a vibratome (VT1000 S, Leica) and collected in PBS as free-oating sections. Sections were rinsed three times in PBS and permeabilized and blocked in PBS containing 0.3% Triton X-100 and 5% normal goat serum (Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature. Sections were then washed in PBS and incubated overnight at 4°C with anti-CDK5 antibody (1:400; Cat# ab40773, Abcam, Cambridge, England), which was detected with an anti-rabbit Alexa Fluor 594 secondary antibody (1:200; Ex: 590 nm, Em: 617 nm; Jackson ImmunoResearch, West Grove, PA, USA). To identify nuclei, sections were counterstained in mounting medium containing DAPI (Sigma-Aldrich). Finally, sections were mounted and examined using a uorescence microscope (Zeiss, Oberkochen, Germany or Leica, Germany).

Electrophysiology
Mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.), and brains were removed. Striatal slices (300 μm) were cut in ice-cold cutting solution [(in mM) 124 NaCl, 2. Patch pipets were prepared from borosilicate glass (Sutter Instrument Company, Novato, CA, USA) using a P-97 Flaming/Brown micropipette puller (Sutter Instrument Company) and had a resistance of 6-8 MΩ when lled with the following intracellular solution [(in mM) 130 CsCl, 10 NaCl, 0.25 CaCl 2 , 2 MgCl 2 , 5 EGTA, 10 HEPES, 10 glucose, 2 Mg-ATP, 0.3 Na 2 -GTP]. The pH of the pipet solution was adjusted to 7.3 with 1 mM CsOH, and osmolarity was adjusted to 285-290 mOsm/L. A low-power objective (4×) was used to identify the DS region, and a 40× water immersion objective (NIR Apo, Nikon, Japan) coupled to an infrared differential interference contrast (IR-DIC) microscope with a uorescence system and a CCD camera was used to visually identify, patch, monitor and record CDK5-KD neurons in the DS. MSNs were identi ed according to previously determined membrane characteristics and ring properties [27]. Recording in normal current-clamp or voltage-clamp modes was performed with a Digidata 1440A digitizer, an Axon 200B ampli er, and Clampex 10.2 software (all from Molecular Devices, San Jose, CA, USA) at room temperature. Fast and slow capacitance compensation was performed after tight-seal (>1 GΩ) formation. During whole-cell recordings, series resistance was monitored and compensated (80-90%) periodically. When the series resistance of a neuron was above 50 GΩ or changed by more than 25%, it was excluded from further analysis. Data were ltered at 2 kHz and acquired at a sampling rate of 10 kHz. Access resistance and leak currents were monitored, and recordings were rejected if these parameters changed signi cantly during data acquisition.
We then examined whether the reduction of CDK5 expression in the DS caused changes in behavioral performance. Locomotor activity was continuously monitored throughout day 14 after LV/Cas9-sgRNA injection. The distance plot of DS-CDK5-KD mice in 5-min blocks revealed severe spontaneous general activity abnormalities (Fig. 2A). Statistical analysis showed that both the total distance traveled [ Fig. 2B; WT, 21965.75 ± 1,827.91 vs DS-CDK5-KD, 13465.04 ± 1265.40; F(2, 21) = 8.54, p = 0.0019] and the total velocity [ Fig. 2C; WT, 5.49 ± 0.34 vs DS-CDK5-KD, 3.74 ± 0.35; F(2, 21) = 6.73, p = 0.006] were signi cantly reduced for DS-CDK5-KD mice. In addition, the activity heatmap revealed that DS-CDK5-KD mice preferred to travel in the peripheral area more than the central area of the arena (Fig. 2D). In line with this, statistical analysis showed that the central distance traveled was signi cantly decreased in DS-Cdk5-KD mice [ Fig. 2E The circadian rhythm of wheel-running activity is altered in DS-CDK5-KD mice To examine the DS-speci c CDK5-driven 24-h night/day rhythms of locomotor activity (free-running), wheel-running activity was continuously monitored during the normal night-day cycle (12/12-h). As shown as Fig. 3A, the WT and DS-CDK5-NC mice sustained normal rhythms of sleep-wake behavior with free movement and foraging during the night (12-h night period) and lower overall activity during the daytime (12-h day period). However, the free-running period of DS-CDK5-KD mice at night was signi cantly shorter than that of WT and DS-CDK5-NC mice. The spontaneous locomotor activity of DS-CDK5-KD mice was only ~20 % of the total activity of control littermates. The activity counts of DS-CDK5-KD mice were signi cantly reduced [ Fig. 3B; WT, 3,549.54 ± 154.20 vs DS-CDK5-KD, 594.77 ± 64.47, F (2, 21) = 38.402, p < 0.001] at night. However, no signi cant differences were observed in activity duration between WT, DS-CDK5-NC, and DS-CDK5-KD mice in 12-h night/12-h day free running (Fig. 3C,). Thus, we calculated the ratio of the dark (α) to light (ρ) phase counts in the night-day cycle. No signi cant differences in α/ρ ratio were observed between WT and DS-CDK5-NC mice. The α/ρ ratio was signi cantly smaller in DS-CDK5-KD mice than in WT mice [ Fig. 3D; WT, 20.34% ± 2.92% vs DS-CDK5-KD, 6.78% ± 1.01%, F (2, 21) = 7.64, p = 0.003]. Corresponding with previous studies, the α/ρ is positively correlated with the circadian period, and abnormal α/ρ indicates a disturbed circadian rhythm. Next, bivariate correlations with behavioral ndings indicated that the α/ρ ratio decrease in wheel running activity signi cantly correlated with other behaviors, such as curling up in the locomotor activity test [ Fig.   3E, r(8) = 0.74, p < 0.001; R 2 = 0.58]. This reduction in overall α/ρ ratio activity in DS-CDK5-KD mice indicates that CDK5 is involved in circadian rhythm behavior in mice. Consistently, bivariate correlations indicated that changes in circadian rhythm may contribute to impaired locomotor activity. CDK5 de ciency causes morphological alterations of MSN dendrites and spines in the DS CDK5 is required for radial neuronal dendrite and spine maintenance [28] and dysregulation of CDK5 dramatically affects striatal-dependent brain function. Here, we show the strati cation of apical MSN morphology in the striatum of WT and DS-CDK5-NC mice and signi cant changes in neuronal morphology in DS-CDK5-KD mice as determined by Golgi staining (Fig. 4A) Fig. 5D; WT, 98.94% ± 3.07% vs DS-CDK5-KD, 87.60% ± 1.38%; F(2, 9) = 3.06, p = 0.097]. However, we found a signi cant decrease in phosphorylation of Tau1 at Thr181 in DS-CDK5-KD mice compared with WT mice, indicating a reduction in the phosphorylation activity of CDK5 at Thr181 [ Fig. 5D; WT, 108.2% ± 3.78% vs DS-CDK5-KD, 87.41% ± 2.85%; F(2, 6) = 14.37, p =0.0052]. These data indicate that downregulation of CDK5 in the DS perturbs dendrite branching and spine formation, as well as the phosphorylation of Tau1 at Thr181 and that CDK5, therefore, plays a key role in synaptic transmission. Together, these results demonstrate that deletion of CDK5 causes morphological disruption and biochemical changes in DS neurons. CDK5 de ciency affects inhibitory synaptic transmission in the DS Deletion of Cdk5 dramatically affects the morphology of MSNs in the DS, which may in turn affect the neuronal signal transduction that underlies behavior. We therefore recorded mEPSCs and sIPSCs using the whole-cell techniques. Electrode placement and action potential properties recorded from MSNs in brain slices are shown in Fig. 6A. The mEPSCs re ect the presynaptic release of neurotransmitter from vesicles (Fig. 6B) CDK5 de ciency in the DS reduces long-range projections to the secondary motor cortex, thalamic nuclei and basolateral amygdala To examine whether Cdk5 knockdown in the DS affects striatal-dependent brain function, we employed anterograde tracing with AAV-hSyn-mCherry to label ber tracts that project from the DS (Fig. 7A). This sensitive neuroanatomical tract tracing technique can be used to visualize neuronal projections, including dendritic arbors [29]. All injections were centered in the DS [coordinates: A/P +1.0 mm, M/L +1.8 mm, D/V -3.2 mm from bregma (Paxinos and Watson, 1986; Franklin and Paxinos, 1997)] and were visualized using uorescence microscopy to check for injection accuracy. Striatal sections obtained after anterograde AAV injection showed that most striatal neurons expressed the mCherry tracer in the DS region (Fig. 7B). Four weeks after AAV-hSyn-mCherry microinjection, whole brain sections were used to trace long-range axonal connections from the DS to the secondary motor cortex (M2), dorsal thalamic nuclei, and the basolateral amygdala (BLA), regions that are closely associated with regulating motor function and circadian rhythm. Fig. 7C, D illustrates the pattern of anterograde labeling observed in the M2, thalamus and BLA in WT, DS-Cdk5-NC and DS-Cdk5-KD mice. In WT and DS-Cdk5-NC mice, the majority of anterogradely labeled cells were located in the M2, thalamic nuclei and BLA, which revealed close contact of all three brain regions with the DS. By contrast, the viral tracers were unevenly and weakly present in the M2, thalamus and BLA of DS-Cdk5-KD mice. Bar graphs of mCherry uorescence in the M2, thalamus and BLA show that uorescence intensities in all three brain areas were signi cantly reduced in DS-Cdk5-KD mice compared with WT mice [mCherry uorescence in the M2 (Fig. 7C): WT, 100.0% ± 6.49% vs DS-Cdk5-KD, 33.91% ± 1.21%; F(2, 6) = 40.68, p =0.003; mCherry uorescence in the thalamus (Fig. 7D): WT,100.0% ±2.78% vs DS-Cdk5-KD,52.97% ± 1.40; F(2, 9) = 40.81, p =0.0003; mCherry uorescence in the BLA (Fig. 7E): WT, 100.1% ± 8.27% vs DS-Cdk5-KD, 41.72% ± 4.37%; F (2, 6) = 18.43, p =0.0027]. These ndings indicate that Cdk5 is important for neural connectivity between the DS and the M2, thalamus and BLA. The DS is the main integration station of the basal ganglia, and CDK5 may have a critical role in maintaining the neural circuits associated with motor function and circadian rhythms in the DS.

Discussion
Striatal dopamine signaling is associated with circadian modulations in the mammalian brain. CDK5 regulation of dopamine neurotransmission in the striatum has been previously evaluated, but the role of CDK5 in circadian regulation is unknown. We therefore used a lentiviral-based CRISPR/Cas9 system to e ciently knockdown the Cdk5 gene in the DS of mice. The DS-CDK5-KD mice exhibited behavioral de cits in locomotor activity and disturbed daily rest/activity cycles, along with dendrite and spine morphological abnormities and impaired basal GABA-mediated sIPSCs in the DS. Furthermore, CDK5 de ciency reduced long range connections from the DS to the M2, thalamus and BLA. These ndings provide insight into the involvement of striatal CDK5 in circadian modulations.
The study of sleep and alertness in neurodegenerative disorders, including PD, is very challenging. In our study, using CRISPR/Cas9-mediated gene editing, we e ciently achieved selective knockout of the Cdk5 gene in the DS of mice (Fig. 1C, D). Fourteen days after viral vector injection, WT and DS-CDK5-NC mice displayed normal locomotor behaviors during the rest/activity cycle. In contrast, DS-CDK5-KD mice exhibited marked reductions in total distance traveled and average moving speed. Moreover, DS-CDK5-KD mice adopted a particular posture (curling up) that coincided with the time at which they were most likely to sleep during the test period (Fig. 2D-F). In addition, we examined the effect of DS-speci c CDK5 knockdown on circadian rhythms of rest/activity behaviors using the running wheel. The circadian system generates a 24-h rhythm of sleep-wake behavior. Control littermates exhibited a circadian rhythm of free-running locomotion, whereas locomotor rhythms in DS-CDK5-KD mice were severely disrupted during the 12-h night period (Fig. 3B, C), with the mice displaying poorly consolidated subjective nighttime activity. The loss of behavioral rhythmicity indicates that striatal CDK5 is largely associated with wakefulness.
CDK5 is an important Ser/Thr protein kinase that participates in actin-binding, synaptic morphology maintenance and postsynaptic organization [30]. CDK5 also regulates the tra cking of synaptic vesicles and neurotransmitter release and contributes to homeostatic scaling [31,32]. Consistently, in DS-CDK5-KD mice, we found changes in dendritic branching and spine formation, as well as altered Cdk5dependent phosphorylation of Tau at Thr181 in the DS region. Proper spine density and morphology, as well as a balance between synaptic excitation and inhibition (E/I balance) is widely regarded to be essential for sleep-wake rhythms [33]. Furthermore, most E/I synapses in the brain are on dendritic spines, which are small protrusions on dendritic shafts that are important for synaptic plasticity [34,35]. Wholecell recording in DS-CDK5-KD mice revealed a speci c decrease in GABA-mediated sIPSCs, while no change was observed in mEPSCs. The GABAergic system, particularly in MSNs, is the major inhibitory neurotransmitter system that underpins E/I balance in the central nervous system. Dysfunction of GABAergic neurotransmission has been implicated in the pathogenesis of numerous behavioral conditions [36][37][38][39]. Therefore, CDK5 may perturb cytoskeletal assembly and spine density, thereby affecting GABAergic synaptic E/I balance in the striatum and reducing the inhibitory output of MSNs ( Fig. 6B-D), in turn resulting in altered sleep-wake rhythms.
Brain functions are mediated by multiple neuronal activities involving highly elaborate and complex synaptic connections. Neurons need to transport organelles, proteins and lipids from the soma to the axon and dendrites and back again to maintain a normal functional state [40,41]. Microtubules act as conduits for both anterograde and retrograde transport of molecules [42,43]. We found here that total Tau expression and the phosphorylation of Tau at Thr181 was signi cantly decreased in DS-CDK5-KD mice (Fig. 5C, D). Tau is highly enriched in neurons and was originally identi ed by its ability to bind to and stabilize microtubules. The equilibrium between Tau phosphorylation and dephosphorylation modulates the stability of the cytoskeleton and synaptic morphology in the normal brain. Consistent with this, we found that CDK5 deletion perturbed the anterograde tra cking of AAV from the DS to the M2, thalamic nuclei and BLA. Nighttime sleep disturbances are common in PD, affecting up to 90% of PD patients [6]. The M2, thalamic nuclei and BLA are closely related with sleep-wake rhythms. The M2 is important for processing and integrating sensorimotor cues, and is involved in motor planning [44]. The thalamus is a critical node that integrates input and output in the central nervous system, and striatalthalamic connections are the foundation of many higher brain functions [45,46]. The BLA is the most important brain region of the circadian clock [47]. Thus, we propose that CDK5 dysfunction affected microtubule equilibrium and transport between the DS and the other brain structures associated with sleep-wake rhythms.
In the current study, we did not examine whether dysfunction of CDK5 is involved in nighttime sleep disturbances in animal models of PD, and this remains to be addressed in future studies. However, in an animal model of PD, in which the neurotoxin, MPTP, is used to selectively induce neurodegeneration of DA neurons in the substantia nigra, higher levels of CDK5 activity are detected. Furthermore, there is increased CDK5-mediated phosphorylation of DARPP-32 at Thr75, which is accompanied by decreased DA-induced phosphorylation of DARPP-32 at Thr34. Accordingly, CDK5 activity is not only required for dendrite and spine maintenance, but also for normal striatal dopamine signaling. The current ndings, therefore, constitute a foundation for extending our understanding of the role of CDK5 in the pathogenesis of sleep-wake rhythm disorders in PD.
Together, our ndings demonstrate a pivotal role of CDK5 in inter-regional connectivity, neurotransmission and motor control in the striatum. Targeted knockout of the Cdk5 gene in the DS produced major changes in dendrite structure at most E/I synapses of MSNs in the striatum. These changes impact communication between the DS and other brain regions, resulting in the dysregulated initiation of sleep-wake rhythm behaviors (summarized in Fig. 8). Collectively, our ndings show that CDK5 in the DS plays a key role in the maintenance of striatal neural circuits associated with motor and circadian rhythm functions.   (E) Pearson correlation between curling up duration in the locomotor activity test with the α/ρ in circadian wheel-running test. Values "R" and "P" are indicated next to linear regression lines. Dashed curves indicate 95% con dence intervals. Data are represented as the mean ± SEM, n = 8 mice per group.