- Open Access
Developmental delay in motor skill acquisition in Niemann-Pick C1 mice reveals abnormal cerebellar morphogenesis
- Paola Caporali†1,
- Francesco Bruno†1,
- Giampiero Palladino1,
- Jessica Dragotto1,
- Laura Petrosini1, 2,
- Franco Mangia1,
- Robert P. Erickson3,
- Sonia Canterini1 and
- Maria Teresa Fiorenza1Email author
© The Author(s). 2016
Received: 29 July 2016
Accepted: 18 August 2016
Published: 1 September 2016
Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disorder caused by defective intracellular trafficking of exogenous cholesterol. Purkinje cell (PC) degeneration is the main sign of cerebellar dysfunction in both NPC1 patients and animal models. It has been recently shown that a significant decrease in Sonic hedgehog (Shh) expression reduces the proliferative potential of granule neuron precursors in the developing cerebellum of Npc1 −/− mice. Pursuing the hypothesis that this developmental defect translates into functional impairments, we have assayed Npc1-deficient pups belonging to the milder mutant mouse strain Npc1 nmf164 for sensorimotor development from postnatal day (PN) 3 to PN21. Npc1 nmf164 / Npc1 nmf164 pups displayed a 2.5-day delay in the acquisition of complex motor abilities compared to wild-type (wt) littermates, in agreement with the significant disorganization of cerebellar cortex cytoarchitecture observed between PN11 and PN15. Compared to wt, Npc1 nmf164 homozygous mice exhibited a poorer morphological differentiation of Bergmann glia (BG), as indicated by thicker radial shafts and less elaborate reticular pattern of lateral processes. Also BG functional development was defective, as indicated by the significant reduction in GLAST and Glutamine synthetase expression. A reduced VGluT2 and GAD65 expression also indicated an overall derangement of the glutamatergic/GABAergic stimulation that PCs receive by climbing/parallel fibers and basket/stellate cells, respectively. Lastly, Npc1-deficiency also affected oligodendrocyte differentiation as indicated by the strong reduction of myelin basic protein. Two sequential 2-hydroxypropyl-β-cyclodextrin administrations at PN4 and PN7 counteract these defects, partially preventing functional impairment of BG and fully restoring the normal patterns of glutamatergic/GABAergic stimulation to PCs.
These findings indicate that in Npc1 nmf164 homozygous mice the derangement of synaptic connectivity and dysmyelination during cerebellar morphogenesis largely anticipate motor deficits that are typically observed during adulthood.
Niemann-Pick type C (NPC) is an inherited lysosomal storage disorder, ultimately fatal and presenting with variable neurovisceral symptoms, age of onset and life span . In spite of broad clinical features, impaired fine motor skills, unsteady gait and balance deficits are the earliest sign of neurological manifestation . The most recent incidence estimate is 1.12 affected patients per 100,000 live births, although this value is likely underestimated because of misdiagnosis . The defect is due to mutations in the genes NPC1 (95 % of cases) or NPC2, encoding for proteins that cooperatively mediate the egress from endosomes/lysosomes of exogenous cholesterol brought to the cells by the low density lipoprotein (LDL)/clathrin-coated pit pathway . The role of NPC1/NPC2 proteins is particularly important in neural cells because cholesterol does not cross the blood–brain barrier once it is fully established after birth , making the adult brain mostly dependent on endogenously-derived cholesterol. Accordingly, cholesterol de novo synthesis occurs in both neurons and astrocytes during early postnatal neurogenesis, thereafter becoming most prominent in astrocytes .
Progressive Purkinje cell (PC) degeneration [1, 7] leading to ataxia, represents the most important neuropathological feature of the disease, although the reason for the selective vulnerability of this neuronal population is currently unknown. Because patients do not apparently show early developmental defects and also because most neuropathological signs appear in Npc1 −/− mice in the juvenile/young adult age, the possibility that early cerebellum development processes are impaired by NPC1-deficiency has mostly been neglected. However, the development and functional maturation of mouse cerebellar cortex is a long-lasting process encompassing the first three postnatal weeks , during which the need for cholesterol is likely to maximize to face the intense glial/neuronal cell proliferation and migration, neurite outgrowth, synaptogenesis and myelin formation. These findings may explain why Npc1 loss-of-function affects the cerebellum more severely compared to other brain regions such as the hippocampus and cortex, whose development is largely completed prior to birth [9, 10]. It has been recently shown that, due to premature exit from the cell cycle, there are a decreased number of granule neurons (GNs) and a 20–25 % reduction in cerebellar lobule size at the end of cerebellar development . This leads to a deficiency of GNs in the Inner Granular Layer (IGL), which may contribute to the later PC degeneration. In line with the robust mitogenic activity Shh exerts on GNs , Shh mRNA levels were found to be significantly reduced at the time of final divisions of GN precursors . Besides GNs, also Bergmann glia (BG) responds to Shh  by differentiating in relationship with PC migration, dendritogenesis, synaptogenesis and maturation , suggesting that Npc1-deficiency also affects the normal pattern of BG differentiation.
Among the animal models of NPC disease, the Npc1 nmf164 mouse is of particular interest because it harbors a single nucleotide substitution (A to G at cDNA bp 3163) causing an aspartate-to-glycine substitution (D1005G) in the cysteine-rich luminal loop, conferring to the NPC1 protein a partial loss of activity as observed in most common human mutations . By assessing the physical and sensorimotor development of pre-weaning Npc1 nmf164 homozygous mice, we have observed a significant delay in the acquisition of complex motor skills compared to wt littermates, which likely indicates an impairment of the cerebellar circuitry functionality. Therefore, we hypothesized that the differentiation of glial cells, including BG and oligodendrocytes, as well as the expression/localization patterns of functional markers of glutamatergic and GABAergic transmission might be altered in Npc1 nmf164 homozygous mice. The evidence we provide in this study, showing that cerebellar morphogenesis is significantly damaged in Npc1 nmf164 homozygous mice substantially confirms our hypothesis.
2-Hydroxypropyl-β-cyclodextrin, a drug promoting cholesterol movement from late endosomes to the metabolically active pool of cholesterol in the cytosol , has been shown to slow the appearance of ataxic symptoms in NPC1 disease mouse [17, 18] and cat models , representing the major treatment currently studied in NPC1 patients. In light of this evidence we assessed whether the administration of this drug rescued the abnormal cerebellar morphogenesis of Npc1 nmf164 mice.
Materials and methods
Animals and treatments
Npc1 nmf164/nmf164 mice with BALB/cJ background (hereafter named Npc1 nmf164 mice) obtained from heterozygous crosses were exposed to a 12 h light–dark cycle, receiving food and water ad libitum. The genotypes of pups were identified by PCR analysis of tail DNA as described . Because a preliminary evaluation ruled out any gender effect on preweaning and adult behavioral performances, male and female mice were grouped together for analyses. Preweaning and adult behavioral performances were analyzed on the same cohorts of 10 Npc1 nmf164 and 10 wt littermates, obtained from 5 litters made of at least 7 pups. Treatment with 2-hydroxypropyl-β-cyclodextrin (hereafter named CD; average degree of substitution of 0.67 of hydroxypropyl groups per glucose unit, MW ~1369 Da, catalog number H-107, Sigma Aldrich, Milan, Italy) was performed by two subsequent subcutaneous injection of either a 20 % solution (w/v; 4000 mg/Kg body weight) of CD in PBS, or plain PBS (sham, control) to 4- and 7-day-old mice Npc1 nmf164 and wt littermates [11, 20]. The effect of CD administration on behavioral performances of preweaning pups was assessed on a cohort of 10 Npc1 nmf164 and 10 wt littermates (5 pups either PBS- or CD-injected/genotype), obtained from 5 litters made of at least 7 pups.
Preweaning behavior assessment
Physical development. The body weight was measured daily in the interval PN3-PN21 and eye opening, fur appearance and incisor eruption were evaluated by visual inspection.
- (b)Development of quadrupedal locomotion. Fluent forward movements with all limbs supporting the whole body and the pelvis elevated were analyzed from PN3 to PN15 by using Ethovision XT software (Noldus, The Netherlands). The pup was placed on a board and video-recorded for 120 s to analyze the following locomotion categories: (i) pivoting, turning movements by broad swipes with forepaws, using only one hindlimb as a pivot and having the pelvis anchored to the ground; (ii) crawling, dragging the body forward or pushing it backward by undulating movements of the trunk and often dragging the hindlimbs in an extended position with foot soles facing upward; (iii) quadrupedal locomotion, smooth and coordinated walking, in which the body is supported in sequence by different numbers of feet in combination, suitable for variegated velocities and without any directional bias. The developmental acquisition of the various locomotion categories was determined as dominant behavior according to the rating scale of Table 1.
Development of swimming performance. The pup was gently released in a glass tank (cm 100 × 50 × 20) filled with warmed (35 °C) water and allowed to swim freely. The parameters swimming direction and limb use were evaluated and scored according to the rating scale of Table 1.
Reflex appearance. (i) Surface righting reflex: the pup was placed gently on its back and the time to turn over on the belly was recorded (allotted time 30 s). (ii) Negative geotaxis: the pup was placed on an inclined (30°) plane with the head pointing downwards and the time to face up to the slope was recorded (allotted time 60 s). (iii) Cliff avoidance: the pup was placed on an edge with forepaws and nose just over the edge and the time to retract itself by backward and/or sideward movements was recorded (allotted time 60 s).
Development of complex motor behaviors. Because the acquisition of complex motor abilities requires the complete maturation of basic reflexes such as the grasping response, which normally appears by the end of the first postnatal week , the development of complex motor behaviors was scored from PN10 on. (i) Ascending a ladder: the pup was placed on a steel ladder (cm 15 × 25, 20 rungs, 1 cm apart, inclination angle 25°) with top leaning against a platform holding littermates. The ability to ascend the ladder within 120 s was evaluated and the day of the first successful performance was recorded. (ii) Crossing a narrow bridge: the pup was placed on the start platform connected by a plywood bridge (40 × 1 × 3 cm) to the goal platform holding littermates. The ability to traverse the bridge within 120 s was evaluated and the day of the first successful crossing was recorded. (iii) Suspension on a wire: the pup was suspended by its forepaws on a wire (2 mm diameter and 50 cm long) extended horizontally between two poles (30 cm high). The suspension time and the first suspension with the 4 limbs (hind limb suspension) were recorded (allotted time 60 s).
Adult behavior assessment
PN30, PN60 and PN90 Npc1 nmf164 and wt littermates were subjected to two daily sessions (morning and afternoon) of the following consecutively administered tests assessing motor behavior : (i) Vertical screen: the mouse was placed on a horizontal wire screen (cm 15x25, wire diameter 2 mm, spaced at 1 cm). The screen was rapidly turned to vertical position with the mouse facing the floor at the lower edge. The latency to turn upward and to climb to the upper edge was measured during 60 s. This test was performed as the first one of the morning session and was not repeated in the afternoon of that day. (ii) Balance beam: the mouse was placed perpendicularly at the center of a horizontal round beam (covered with paper tape, outer diameter 2 cm, length 1 m, divided into 10 sections and placed 50 cm above a padded surface). The retention time and the number of beam sections crossed during 180 s were recorded and the results of morning and afternoon trials were averaged. (iii) Coat hanger: the mouse was suspended in the middle of the horizontal bar of a coat hanger (diameter 3 mm, length 35 cm, placed 30 cm above a padded surface) with its forepaws. The body position of the animal was observed for 60 s and scored as follows: 0, a fall within 10 s; 1, grasping the hanger with one limb; 2, grasping the hanger with two limbs; 3, grasping the hanger with three limbs; 4, grasping the hanger with four limbs; 5, actively escaping to the end of the bar. The values of morning and afternoon trials were averaged.
These tests were selected because they were similar to those we had exploited in behavioral analyses of preweaning pups in terms of functions evaluated and experimental setting.
Western blot assays
Santa Cruz Biotechnolgy, Santa Cruz, CA, USA; #sc-33673
Anti-EAAT1 or GLAST
AbCam, Cambridge, UK; #ab416
Thermo Fisher Scientific, Rockford, IL; USA; #PA5-25653
Sigma-Aldrich Inc., St. Louis, MO, USA; #M3821
Horseradish peroxidase-conjugated goat anti-rabbit IgG
Thermo Fisher Scientific; #32460
Horseradish peroxidase-conjugated goat anti-mouse IgG
Thermo Fisher Scientific; #32430
Horseradish peroxidase-conjugated goat anti-mouse IgG2a
Santa Cruz Biotechnolgy; #sc-2061
Biotinylated goat anti-rabbit IgG
Vector Laboratories, Burlingame, CA; #PK-6101
Biotinyted goat anti-mouse IgG
Vector Laboratories; #PK-6102
PN15 Npc1 nmf164 and wt littermates (4 mice/genotype) were deeply anaesthetized by intraperitoneal injection of a mixture of xylazine (20 mg/Kg) and ketamine (34 mg/Kg) and then transcardially perfused with 4 % PFA in 0.1 M PBS. Brains were removed and post-fixed overnight at 4 °C in 4 % PFA. For MBP detection, PFA-fixed brains were dehydrated, embedded in Paraplast Tissue Embedding Medium (Leica Biosystems, Milan, Italy) and serially sectioned (slice thickness 8 μm). Sagittal sections were mounted on X-tra Adhesive glass slides (Leica Biosystems), de-waxed with xylene, rehydrated and washed in PBS. The detection of other glial and neuronal cell markers was performed on cryosections. To this end, fixed brains were cryoprotected with sucrose (30 %, w/v, in PBS), embedded in FSC22 Clear R Frozen Section Compound (Leica Biosystems) and serially sectioned (slice thickness 8 μm) using a Leica CM 1900 cryostat. For GLAST detection, cryosections were subjected to 20 min fixation in acetone (−20 °C), which significantly improved antigen detection . Paraffin sections and cryosections were then processed for epitope unmasking and endogenous peroxidases inactivation. For antigen unmasking, sections were incubated (5 min × 2) in 10 mM sodium citrate, pH 6.0 in a microwave oven and then in 0.3 % H2O2 for 15 min at RT to inactivate endogenous peroxidases. A 2 h incubation in a blocking solution made of 0.5 % BSA in PBS preceded the incubation of sections with anti-GLAST and anti-MBP antibodies. For the detection of VGlut2, GFAP, Glutamine synthetase and GAD65 the blocking solution was supplemented with 0.1 % Triton X-100. The incubation of sections with primary antibodies lasted approximately 18 h at 4 °C and was followed by several washes in PBS before exposure to the appropriate secondary antibody (see Table 2 for details). Antibody-antigen complexes were revealed with Vectastain Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA, USA) followed by DAB Peroxidase Substrate Kit (Vector Laboratories Inc.), according to manufacturer’s instructions. Immunodetection specificity was assessed by omitting the primary antibody. Images were obtained using a Zeiss Axioplan microscope equipped with a Sony nex-3 N mirror-less camera (Sony Europe Limited, Milano, Italy) and processed using ImageJ NIH software (National Institutes of Health, Bethesda, MD).
VGluT2- and GAD65-positive puncta were quantitated in 3–4 sagittal sections of 4 mice/genotype as previously described [26, 27], with slight modifications. Images were acquired using a Zeiss Axioplan microscope at 100X magnification (Neofluar, 0.7–1.30) and a Sony nex-3 N mirror-less camera. For each antibody, at least 8 image fields of lobule II and lobule X were acquired along the molecular layer starting from the pial surface. The abundance of VGluT2- and GAD65-positive puncta was determined in regions of interest (ROI) of 6500 μm2 and 3200 μm2, respectively, randomly selected in outer and inner molecular layers by the “cell counter” function of ImageJ NIH software. The number of GAD65-positive puncta around the PC’s soma was also determined. Only VGluT2- and GAD65-positive puncta having a high-to-moderate staining and a diameter of 0.3–1.3 μm were counted. All determinations were performed blindly and independently by two investigators. Because no significant difference was observed between counts of lobule II and lobule X microscopic fields of wt or Npc1 nmf164 mice, data were pooled.
Statistical analyses were performed with STATISTICA 8 (StatSoft) software. Data were first tested for normality (Wilk-Shapiro’s test) and homoscedasticity (Levene’s test), and then analyzed by unpaired two-tailed Student’s t test or two-way ANOVAs for independent (genotype, treatment) and repeated (age) measures, followed by Bonferroni’s post-hoc test. When data did not fully meet parametric assumptions or were ordinal (locomotion and swimming measures), comparisons between groups were performed by Mann-Whitney’s U test. To control for alpha inflation, i.e. the proportion of type I errors among all rejected null hypotheses, the False Discovery Rate (FDR) was set to 0.05 and estimated through a bootstrap procedure . Differences were considered to be significant at the p ≤ 0.01 level.
Npc1 nmf164 mice show a delay in the acquisition of complex motor skills requiring fine motor coordination and balance
Statistical analysis outputs of quadrupedal locomotion and swimming performance development in Npc1 nmf164 and wt littermatesa
Z = 1.00; p = 0.74
Z = 0.00; p = 1.00
Z = 0.00; p = 1.00
Z = −1.00; p = 0.74
Z = 0.00; p = 1.00
Z = 0.00; p = 1.00
Z = 1.00; p = 0.74
Z = 1.90; p = 0.14
Z = 0.89; p = 0.48
Z = −1.00; p = 0.74
Z = 0.00; p = 1.00
Z = 1.09; p = 0.48
Z = −0.61; p = 0.74
Z = 0.00; p = 1.00
Z = 1.00; p = 0.74
Z = 0.97; p = 0.53
Z = 0.00; p = 1.00
Z = 0.00; p = 1.00
Z = −0.59; p = 0.63
Z = 0.00; p = 1.00
Z = 0.00; p = 1.00
Z = 0.00; p = 1.00
Z = 0.00; p = 1.00
Z = −1.00; p = 0.74
Z = −1.09; p = 0.48
Z = −1.09; p = 0.48
Z = −1.00; p = 0.74
Z = 1.37; p = 0.28
Z = 0.89; p = 0.48
Z = 0.00; p = 1.03
Z = −1.13; p = 0.44
Z = 1.83; p = 0.28
Z = 0.89; p = 0.48
Z = −1.45; p = 0.48
Z = 0.00; p = 1.00
Z = 2.18; p = 0.14
Z = 0.00; p = 1.00
Z = 0.00; p = 1.00
Z = 1.00; p = 0.74
In the mouse, complex motor abilities requiring fine limb coordination, balance and muscle strength are normally acquired by the end of the second postnatal week. Three tasks (ascending a ladder, crossing a narrow bridge and suspension on a wire) allowed us to differentiate the contribution of motor coordination and balance from that of grip and muscle strength. Npc1 nmf164 pups acquired these abilities with a significant delay compared to wt littermates (Fig. 2f). Indeed, whereas wt pups crossed the narrow bridge in its entire length and hanged on the wire with four limbs after PN14, Npc1 nmf164 mice crossed the bridge only at PN17 (main effect of genotype: Z = −2.54, p = 0.01) and developed the four-limb hanging ability at PN18 (main effect of genotype: Z = −2.98, p = 0.004). In contrast, grip ability and muscle strength developed similarly in Npc1 nmf164 and wt littermates, as shown by their similar ability to ascend the ladder after PN15 (main effect of genotype: Z = 0.27, p = 0.80) and to hang on the wire for a longer time with increasing age (main effect of genotype: F 1,18 = 1.09, p = 0.31; main effect of age: F 10,180 = 3.23, p = 0.0008; interaction between genotype and age: F 10,180 = 0.20, p = 0.99).
The possibility of evaluating the efficacy of CD to rescue the developmental delay in motor skills acquisition of Npc1 nmf164 and wt littermates was hampered by the hyperactivity of mouse pups elicited by the injection per se. Both CD-treated and sham group pups, regardless of genotype resisted our attempts to perform motor behavior assessments.
Motor deficits of Npc1 nmf164 mice become more severe in adulthood
To fully characterize motor phenotype in adults, PN30, PN60 and PN90 Npc1 nmf164 and wt littermates were subjected to a battery of tests including Vertical screen, Balance beam, and Coat hanger.
The Balance beam test (similar to crossing a narrow bridge) measures fine motor coordination and balance (Fig. 3b). When placed on an elevated round beam, Npc1 nmf164 mice crossed significantly fewer beam sections than wt mice did and significantly fewer sections as days went by (main effect of genotype: F 1,18 = 34.92, p = 0.00001; main effect of age: F 2,36 = 5.08, p = 0.01; interaction between genotype and age: F 2,36 = 4.09, p = 0.03). Moreover Npc1 nmf164 mice did not differ from wt until PN90 in terms of retention time (main effect of genotype: F 1,18 = 54.28, p < 0.00001; main effect of age: F 2,36 = 6.48, p = 0.004; interaction between genotype and age: F 2,36 = 6.01, p = 0.006).
The Coat hanger test (similar to suspending on a wire) further characterizes motor coordination by providing an “agility score” (Fig. 3c). Npc1 nmf164 mice obtained scores lower than those of wt mice when suspended on the coat hanger. In fact, while wt mice rapidly escaped to the bar end, Npc1 nmf164 mice did not progress to the end of the bar although they were able to grasp the bar with four limbs (main effect of genotype: F 1,18 = 18.81, p = 0.0004; main effect of age: F 2,36 = 3.80, p = 0.03; interaction between genotype and age: F 2,36 = 2.30, p = 0.11).
The possibility that body weight influenced motor behavior was routinely checked before all behavioral evaluations (Fig. 3d). Body weight of Npc1 nmf164 and wt mice did not differ at PN30 and PN60, while it significantly decreased in PN90 Npc1 nmf164 mice, as previously described  (main effect of genotype: F 1,18 = 13.35, p = 0.002; main effect of age: F 2,36 = 125.40, p < 0.00001; interaction between genotype and age: F 2,36 = 22.26, p < 0.00001).
Bergmann glia morphogenesis and functions are defective in Npc1 nmf164 mice
Our analysis of the gross morphology of PN15 Npc1 nmf164 mouse cerebellum showed that the number of GNs forming the external granule layer was significantly reduced compared to age-matched wt mice (Additional file 1 and Additional file 2: Figure S1A-B), suggesting a defective proliferation of GN precursors similar to that previously observed in Npc1 −/− mice . The quantification of cells incorporating BrdU (Additional file 2: Figure S1C-D) confirmed this possibility and prompted us to further analyze the cerebellar morphogenesis of these mice.
Purkinje cells of Npc1 nmf164 mice display a reduced number of glutamatergic and GABAergic inputs
Npc1 nmf164 mice display defective myelin maturation
Immunohistochemistry of PN15 cerebellar sections fully confirmed the impairment of myelin formation in Npc1 nmf164 mice, showing a significant reduction of MBP immunostaining at the level of PC axons and white matter (Fig. 8b). To further characterize this defect, similar analyses were also performed on cerebral cortex and corpus callosum, i.e. brain areas in which individual oligodendrocytes are more easily detected (Fig. 8c). Based on these analyses, the dysmyelination of Npc1 nmf164 mice appeared to be associated with poor oligodendrocyte differentiation, as indicated by the reduced length of the processes that typically radiate from oligodendrocyte soma. Accordingly, the MBP immunostaining of cerebral cortex and corpus callosum was strongly reduced, in agreement with previous observations .
CD treatment partially rescues the abnormal development of glial and neuronal cells in Npc1 nmf164 mice
Statistical analysis outputs on Western blot assays of wt and Npc1 nmf164 littermates either sham- or CD-treated
Genotype x treatment
F (1,12) = 29.35, p = 0.0001
F (1,12) = 52.17, p < 0.0001
F (1,12) = 0.70, p = 0.42
F (1,12) = 199.21, p < 0.0001
F (1,12) = 152.93, p < 0.0001
F (1,12) = 8.78, p = 0.01
F (1,12) = 22.77, p = 0.0005
F (1,12) = 95.83, p < 0.0001
F (1,12) = 9.63, p = 0.01
F (1,12) = 12.85, p = 0.004
F (1,12) = 12.51, p = 0.004
F (1,12) = 11.48, p = 0.005
F (1,12) = 0.83, p = 0.38
F (1,12) = 5.47, p = 0.04
F (1,12) = 26.21, p = 0.0003
F (1,12) = 13.53, p = 0.003
F (1,12) = 22.02, p = 0.0005
F (1,12) = 21.89, p = 0.0005
F (1,12) = 0.14, p = 0.71
F (1,12) = 3.37, p = 0.09
F (1,12) = 74.51, p < 0.0001
F (1,12) = 0.04, p = 0.84
F (1,12) = 5.60, p = 0.04
F (1,12) = 24.13, p = 0.0004
F (1,12) = 1.55, p = 0.24
F (1,12) = 47.36, p < 0.0001
F (1,12) = 92.86, p < 0.0001
We have shown that Npc1 nmf164 mice acquire fine motor coordination and balance as well as complex abilities depending on cerebellar maturation [48, 49] with a significant delay compared to wt littermates, in spite of their normal physical and postural development. An overall disturbance of cerebellar morphogenesis underscores this phenotype, emphasizing the relevance of exogenous cholesterol uptake and Npc1-mediated intracellular trafficking for proper cerebellar development. The presence of a developmental delay instead of a more severe deficit of these abilities is likely explained by the greater plasticity of developing cerebellum and/or the availability of cholesterol of neuronal origin before the full shift to astrocyte-derived cholesterol, which compensates the deficit of Npc1 function.
The normal appearance of sensorimotor reflexes and locomotion development of Npc1 nmf164 pups within the first two postnatal weeks indicate that vestibular, tactile, and proprioceptive systems; descending motor pathways; and brain stem-spinal networks [30, 49] are not apparently affected by Npc1-deficiency. Conversely, the domain of complex motor abilities is damaged by Npc1-deficiency because they are also prematurely lost in the adulthood. In fact, motor coordination and balance are more severely impaired than grip capacity and muscle strength as early as at PN30 and these motor defects thereafter translate to severe ataxia, because of the massive PC degeneration [15, 24].
The acquisition of complex motor abilities depends on proper sequencing and coordination of motor outputs. These are prominent properties of cerebellar circuitry , consisting of several functional modules that allow the real-time control of movements and the long-term changes underling motor learning, by finely regulated signal generation and flow that ultimately converge on PCs. The inhibitory activity of PCs is dynamically orchestrated at the level of both dendritic shafts and cell body by a number of excitatory and inhibitory neurons, while PCs in turn modulate the excitability of deep cerebellar nuclei. Therefore, an altered pattern of synaptic inputs to PCs may affect the timing of their firing and finally result in behavior abnormalities. For example, it was recently demonstrated that an altered GN development results in impaired motor coordination [50, 51]. Similar features were also reported as a consequence of a defective development of BG processes [52–54], likely because correct BG development is crucial for cerebellar cytoarchitecture and function . Conversely, a precocious BG and PC maturation is associated with an earlier acquisition of motor abilities in young and improved motor learning and coordination in adult mice . In light of these findings, it is possible that the developmental delay in the acquisition of complex motor skills we have observed in Npc1 nmf164 pups results from a derangement of synaptic inputs to PCs.
Indeed, we found several developmental anomalies that impinge on the functionality of PCs, suggesting the possibility that the selective vulnerability of these cells represents the final outcome of a number of developmental defects in glial and neuronal cells forming the ordered pattern of cell-to-cell interaction and synaptic connectivity of cerebellar cortex . For instance, the abnormal BG differentiation (thicker radial shafts and a less elaborate reticular pattern of lateral processes) we observe in PN15 Npc1 nmf164 mice may be particularly relevant. In fact, BG processes organize the compartmentalization pattern of synaptic inputs that reach PCs , playing a prominent role in the differential guidance and targeting of basket and stellate cell axons . In addition, BG processes finely regulate cerebellar synaptic activity  by almost completely enwrapping the synapses that parallel and climbing fibers establish with PCs [14, 36].
The glutamate transporter GLAST finely regulates PC firing at BG perisynaptic processes by preventing glutamate spillover between adjacent PCs  and maintaining the one-to-one functional relationship between climbing fibers and PCs that is crucial for cerebellar control of motor function [54, 58, 59]. Because the glutamate recovered by GLAST is metabolized to glutamine by Glutamine synthetase , the reduced expression of GLAST and Glutamine synthetase in Npc1 nmf164 mice is in line with the proposal that GLAST is a limiting factor in glutamate synthesis . Also, GFAP plays a key role in astrocyte-neuron interactions, by modulating the trafficking and function of astrocytic and neuronal glutamate transporters, as well as glutamine production . All together these findings suggest that the abnormal morphological differentiation of BG affects the functional specialization of their processes, as also indicated by the reduced expression of GLAST and Glutamine synthetase. In addition to BG, a decrease of GLAST and Glutamine synthetase expression is likely to occur in astrocytes, the functional impairment of which is indicated by astrocytosis typically displayed by Npc1 nmf164 cerebella. In this regard it is worth noting that, in Npc1-deficient mice, astrocytosis is consistently accompanied by microglia activation [15, 63, 64] and the down regulation of GLAST has been found to correlate with the release of inflammatory cytokines by activated microglia .
Npc1 is also abundant in the recycling endosomes of presynaptic terminals. In fact, Npc1-deficiency results in morphological, biochemical and functional modification of both excitatory and inhibitory presynaptic terminals and synaptic vesicle turnover . This may explain the reduction of both excitatory and inhibitory inputs received by PCs, as indicated by the significant reduction of VGluT2 and GAD65 puncta. This imbalance of synaptic inputs associated with Npc1-deficiency is in agreement with previous findings showing a decrease of synaptic inputs to PCs in co-cultures of Npc1-deficient neurons and glial cells . The lower number of GNs that are generated in the cerebellum of Npc1 nmf164 mice likely contributes to the reduction in glutamatergic inputs, as indicated by our finding that VGluT2 puncta reduction is prominent in the outer molecular layer, which is mostly made of GN axons. On the other hand, the GN reduction may also impinge on the full differentiation of basket/stellate interneurons, which, among other intrinsic genetic programs and extracellular cues, depends on connectivity with GN axons [68, 69]. Along the same line, the lower number of GNs may also be responsible for the abnormal differentiation of BG processes in Npc1 nmf164 mice, because the glutamate released by parallel fibers modulates the degree of BG perisynaptic envelopment acting through calcium-permeable AMPA receptors .
Defective oligodendrocyte maturation likely underlines the overall reduction in MBP we have observed in Npc1 nmf164 cerebellum, in line with previous studies showing dysmyelination in both NPC patients and Npc1 −/− mice [43, 70]. Moreover, the decreased MBP expression not only affected the 18.5 kDa (specific to mature oligodendrocytes), but also the 17.5 kDa and 21.5 kDa isoforms (specific to developing oligodendrocytes), indicating that oligodendrocyte differentiation per se is also impaired in Npc1-deficient mice. Although Npc1 deletion in neurons triggers the block of oligodendrocyte maturation and thus leads to a subsequent failure of myelin formation , the exogenous cholesterol uptake by oligodendrocytes coupled to Npc1-mediated intracellular trafficking is also relevant for the formation of myelin sheaths [40, 44]. Accordingly, oligodendrocyte ablation during the first postnatal weeks gives rise to ataxia and motor deficits in the mouse [39, 71]. By showing that a significant myelin reduction is prominent at the level of PC axons, our results further corroborate the convergence of various Npc1 deficiency-dependent abnormalities on PC functionality.
Present findings also demonstrate that early postnatal CD treatment effectively re-wires developmental trajectories, by partly rescuing the defective cerebellar morphogenesis and thus explaining the well-established beneficial effect a single CD administration to PN7 Npc1 −/− mice has in rescuing lysosomal cholesterol accumulation and slowing down the appearance of ataxic symptoms [17–19, 72, 73]. In fact, this treatment is particularly timely because cerebellar morphogenesis maximizes the need for cholesterol, making the exogenous LDL-uptaken cholesterol a rate-limiting factor for neurons [74, 75]. Noteworthy, CD administration didn’t rescue either GFAP hyper- or GLAST hypo-expression of Npc1 nmf164 mice, although it fully rescued Glutamine synthetase levels in these mice. Because Glutamine synthetase is mainly expressed by astrocytes, this observation rules out the possibility that astrocytes are not influenced by CD, making this issue worthy of further investigation, also in light of the ability of CD administration to influence GFAP and GLAST expression of wt mice.
In conclusion, we correlate the delay of complex motor skills acquisition by Npc1 nmf164 mice to a number of glial cell differentiation anomalies and derangement of synaptic input to PCs. We believe that these findings are relevant because: i) delineate a novel perspective to explain the selective Purkinje cell vulnerability in NPC1 mouse models and patients; and, ii) emphasize the need of early diagnosis to secure the best treatment efficacy in patients.
The financial supports of Telethon Foundation - Italy (grant no. GGP13183 to M.T.F.) and the Ateneo La Sapienza (C26V127RC3) are gratefully acknowledged.
PC designed and performed behavioral analyses and drafted the manuscript; FB, GP and JD performed immunohistochemistry, western blot analyses and interpreted data; LP supervised and interpreted behavioral studies; FM and RPE contributed with advise, discussion and manuscript editing; SC supervised experimental work, analyzed and discussed data; MTF conceived the study, interpreted data and drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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