deficiency cerebellar postnatal development of microglia and climbing fiber refinement in a mouse model of Niemann-Pick Type C disease.

Genetic deficiency of Npc1 impairs postnatal development of microglia and climbing fiber synaptic pruning in the mouse cerebellum. Abstract Little is known about the effects of NPC1 deficiency in brain development and if they contribute to neurodegeneration in Niemann-Pick Type C disease. Since cerebellar Purkinje cells die early and to a higher extent in NPC, here we analyzed the effect of NPC1 deficiency in microglia and climbing fiber synaptic refinement during cerebellar postnatal development using the Npc1 nmf164 mouse. Our analysis revealed that NPC1 deficiency leads to early phenotypic changes in microglia that are not associated with an innate immune response. However, the lack of NPC1 in Npc1 nmf164 mice significantly affected the early development of microglia by delaying the radial migration, increasing the proliferation and impairing the differentiation of microglia precursor cells during postnatal development. Additionally, increased phagocytic activity of differentiating microglia was found at the end of the second postnatal week in Npc1 nmf164 mice. Moreover, significant Climbing-fiber (CF) synaptic refinement deficits along with an increased engulfment of CF synaptic elements by microglia were found in Npc1 nmf164 mice, suggesting that profound developmental defects in microglia and synaptic connectivity precede and predispose Purkinje cells to early neurodegeneration in NPC. Our results suggest These results indicate that alters microglia.


Introduction
Niemann-Pick Type C disease (NPC) is a recessive genetic lysosomal storage disease caused by mutations in the NPC1 or NPC2 proteins, important transporters of cholesterol from endosomes and lysosomes (Patterson, 1993).
Accumulation of cholesterol inside these intracellular organelles leads to progressive neurodegeneration, dementia, and death in children. Developmental regression, ataxia, and cognitive impairment are found among the symptomatology of NPC.
Although the average age of diagnosis is 10yrs, 50% of the NPC patients are diagnosed before age 7 and die before 12.5yrs of age. However, the average age of death is 16yrs, indicating that NPC onset and severity are variable but progressive (Garver et al., 2007). In fact, the nature and severity of neurological symptoms in NPC are directly correlated to the onset of the disease. Infantile manifestations of NPC include delay in motor milestones, gait problems, clumsiness and speech delay, while symptomatic manifestations of juvenile and adult-onset of NPC include learning deficits, ataxia, dystonia and psychiatric symptoms (Mengel et al., 2013).
Questions remain as to how the deficiency of the NPC1 protein perturbs developmental processes in the brain that could contribute to the early development of dementia and neurodegeneration.
Studies in mice and cats have revealed that Purkinje cells (PCs) are particularly hypersensitive to the loss of the NPC1 protein; in NPC these cells degenerate earliest and to a greater severity than other neurons in the brain (Vanier and Millat, 2003). This early degeneration of cerebellar PCs contributes to the development of early neurological symptoms such as clumsiness, gait defects and ataxia in both the human disease and animal models. Identifying the timeline and nature of pathological changes at the cellular and molecular level in the cerebellum Development • Accepted manuscript caused by mutations in Npc1 is critical to understand the mechanisms underlying the early dysfunction and degeneration of PCs. Interestingly, recent studies have shown that genetic inactivation of reactive microglia in Npc1 -/mice reduces neurological impairment and increases their life span (Cougnoux et al., 2018). We recently found that engulfment and phagocytosis of dendrites by activated microglia occur early and precede PC loss in NPC (Kavetsky et al., 2019), demonstrating that activated microglia contribute to PC degeneration in NPC.
Although NPC is a childhood disorder, little is known about the effects of NPC1 deficiency on brain development. Moreover, how developmental deficits precede and contribute to neurodegeneration in NPC is not completely understood.
Developmental delay in motor skill acquisition and significant reductions in synaptic and myelin proteins were reported in the postnatal Npc1 nmf164 mouse (Caporali et al., 2016). In contrast to mice with complete deletion of the Npc1 gene, the Npc1 nmf164 mutant mice present a late onset and slower disease progression where severe motor deficits and neurodegeneration are evident at a young adult stage (Maue et al., 2012). Since no degeneration of PC and no severe motor deficits are found at early developmental stages, the Npc1 nmf164 mouse is an ideal model to study potential "silent" cerebellar developmental defects and behavioral changes caused by NPC1 deficiency that precede degeneration of PC. Contrary to most common late-onset dementias that are age-associated, the early-childhood onset of neurological manifestations in NPC and other lysosomal storage disorders (LSD), indicates the potential disruption of neurodevelopmental processes. The impacts of disrupted cholesterol trafficking by NPC1 deficiency in neural cells development is unknown, leading to a poor understanding of the origin and preclinical mechanisms that lead to childhood dementia. Understanding the mechanisms by which NPC1 Development • Accepted manuscript deficiency affects neural cells during development not only will expand our current knowledge of brain-behavior developmental processes, but also will provide potential therapeutic avenues to identify and delay the progression of NPC and other childhood dementias (Ford et al., 1951;Shapiro E.G., 1994).
Previous work in our laboratory showed significant changes in microglia number, morphology and phagosome content in the cerebellum of Npc1 nmf164 mice at post-weaning age and before PC degeneration (Kavetsky et al., 2019). Interestingly, the migration, proliferation and differentiation of cerebellar microglia occur postnatally along with the development and differentiation of cerebellar neurons. In the brain, microglia play important roles during normal development, including the clearance of apoptotic cells and the elimination of redundant synapses. Importantly, when compared to microglia from other regions of the brain, normal cerebellar microglia strongly display cellular and gene expression patterns mostly associated with cell clearance (Ayata et al., 2018), suggesting that cerebellar microglia are more phagocytic. To determine the impact of NPC1 deficiency in cerebellar microglia during postnatal development, different stages of microglia development such as migration, proliferation and differentiation were examined at the corresponding postnatal age. The findings of this study suggest that NPC1 deficiency not only affects the different phases of microglia development, but also increases phagocytosis activity in these cells that promotes and amplifies profound synaptic defects during the postnatal development of the cerebellum.

Results
Early changes in microglia are not the result of an innate immune response in the cerebellum of post-weaning Npc1 nmf164 mice.
Since changes in Npc1 nmf164 microglia are evident at P30 (Kavetsky et al., 2019, Fig.1A), we proceeded to test if genes associated with an innate immune response were upregulated in the cerebellum of P30 Npc1 nmf164 mice. A PCR array plate containing primers for ~92 genes associated with the mouse innate-immune response was used. RNA from the cerebellum of P90 Npc1 nmf164 mice was used as a positive control since at this age a severe loss of PCs is detected and the changes in microglia morphology and proliferation are more remarkable than at P30 (Fig. 1A) (Kavetsky et al., 2019). As expected, several genes including cytokines (Lif,Tnf,Csf3,Il1a,Ccl2,and Ccl3), endothelial-inflammatory genes (Sele and Vcam1), T-cell associated genes (Gzmb,Cd3e,Cd28,Stat4,PRF1,and Tnfrsf18) and other proinflammatory molecules (C3 and Ptgs2) were significantly increased (>5 fold) in the cerebellum of P90 Npc1 nmf164 mice when compared to WT mice (Fig. 1B).
However, no significant expression changes were found in the cerebellum of P30 Npc1 nmf164 mice when compared to WT mice (Fig. 1B), suggesting that microglia changes at that early age are not the consequence of an immune or inflammatory response in Npc1 nmf164 mice. To determine if microglia changes at P30 were associated with effects of NPC1 deficiency on microglia development, a comprehensive analysis of cerebellar and microglia postnatal development was performed.

Development • Accepted manuscript
Early radial migration of microglial precursors into the developing cerebellar cortex is reduced in Npc1 nmf164 mice.
In the mouse, most of the cerebellar development occurs postnatally. Therefore, at early postnatal stages such as P4, only round and ameboid IBA1 + microglial precursors were observed in the developing cerebellar medulla (CbM) while ramifying microglia were already found at this stage in the cerebral cortex ( Fig. 2A).
As described by others (Ashwell, 1990;Cuadros et al., 1997;Mosser et al., 2017), in the P4 WT cerebellum microglial precursors were concentrated in the developing CbM and migrating tangentially towards the primitive cerebellar folia following tomato lectin + blood vessels (Fig. 2B). Also, at this early postnatal stage, abundant microglial precursors were observed at the pial surface (PS) of the meninges (Fig.   2B). The beginning of the radial migration of microglial precursors into the different regions of the cerebellar cortex that include the inner granule layer (IGL), Purkinje cell layer (PCL) and external granule layer (EGL), is expected to occur at this early stage. Quantitative analysis of the density of IBA1 + microglial precursors in the CbM revealed no differences between P4 WT and Npc1 nmf164 mice ( Fig. 2B and C).
However, a significant reduction in the number of IBA1 + microglial precursors reaching the IGL and circulating the PS was found in Npc1 nmf164 mice when compared to WT mice ( Fig. 2B and D). At this early postnatal stage, very few IBA1 + microglial precursors have reached the PCL, and the EGL is completely devoid of them as reported by others (Cuadros et al., 1997;Nakayama et al., 2018). No changes in vascularization (tomato lectin + blood vessels) were observed between the P4 WT and Npc1 nmf164 mice (Fig. 2B, Fig. S1).
Proliferative activity in CbM microglial precursors was detected in both P4 WT and Npc1 nmf164 mice, but a significantly higher number and percentage of these CbM Development • Accepted manuscript KI67 + /IBA1 + microglial precursors were found in Npc1 nmf164 mice ( Fig. 2E-G).
Meanwhile, proliferative activity in microglial precursors at the IGL, PCL, and PS was very low in P4 WT and Npc1 nmf164 mice, but a further significant reduction in proliferative microglial precursors was found in Npc1 nmf164 mice ( Fig. 2H and I), mostly because of the delayed migration of these cells in the Npc1 nmf164 mice.
Overall, these results suggest that NPC1 deficiency affects the ability of microglial precursors to migrate radially while increasing their proliferation during early postnatal cerebellar development.

Increased density of precursor and maturing microglia in the developing cerebellum of P10 and P14 Npc1 nmf164 mice
Studies have shown that the proliferative activity of microglial precursors in the developing cerebellar CbM and folias white matter region (WMR) start at birth and peak at P7 in normal mice (Ashwell, 1990;Li et al., 2019). In fact, an abundant subpopulation of microglia in the WMR region was recently identified as proliferativeregion-associated microglia (PAM) (Li et al., 2019). When we examined the cerebellum of P10 WT and Npc1 nmf164 mice, we found that the volume occupied by IBA1 + microglia in the WMR (axonal tracts) was significantly higher in Npc1 nmf164 mice than in WT mice ( Fig. 3A-B and D). However, the percentage of proliferative microglia in this region, as assessed by KI67 immunostaining, was similar between WT and Npc1 nmf164 mice ( Fig. 3B and E). Similarly, the density of IBA1 + microglia in the PCL and molecular layer (ML) was significantly increased in Npc1 nmf164 mice without significant changes in KI67 immunoreactivity when compared to WT mice ( Fig. 3C and F-G). In addition, microglia in the cerebellar cortex of WT and Npc1 nmf164 mice were evidently maturing and ramifying (Fig. 3C), while the microglia Development • Accepted manuscript in the WMR were ameboid shape and less ramified, an indication of a more immature cell (Fig. 3B). Since no differences in KI67 immunoreactivity at the cerebellar WMR were observed between P10 WT and Npc1 nmf164 mice, our results suggest that an increased number of microglia was produced in the cerebellar WMR of Npc1 nmf164 mice prior to the P10 stage (P4-P7) as reported by others (Li et al., 2019). To test this possibility, P10 WT and Npc1 nmf164 cerebellar slices were immunostained with CLEC7A (Fig. 4), a specific marker for PAM cells, which proliferation rate in WT mice is significantly reduced after peaking at P7 (Li et al., 2019). Interestingly, a significant higher number of CLEC7A + clusters were observed in Npc1 nmf164 developing WMRs ( Fig. 4A and C). Also, the area of these clusters with CLEC7A + (Fig. 4D) and IBA1 + cells (Fig. 4E), as well as the fraction of the IBA1 + area that was CLEC7A + (Fig. 4F), were significantly larger in Npc1 nmf164 mice when compared to WT. The majority of P10 CLEC7A + microglial precursors lacked processes and were primarily ameboid shape in both WT and Npc1 nmf164 mice ( CLEC7A + /IBA1 + cells were located (Fig. 4K, blood vessels staining is artifactual). We found that MBP intensity inside the CLEC7A + clusters tended to be decreased in Development • Accepted manuscript Npc1 nmf164 mice, but due to variability in WT mice the result was not statistically significant ( Fig. 4L).
At P14, the density of IBA1 + microglia was also significantly higher in the PCL/ML of Npc1 nmf164 mice when compared to WT mice ( Fig. 5A-C). However, the levels of IBA1 + /KI67 + cells in P14 WT and Npc1 nmf164 mice were very low in the cerebellar cortex layers ( Fig. 5B and D). Overall, our results suggest that the increased proliferation of WMR microglial precursors in Npc1 nmf164 mice leads to an increased density of microglia in the cerebellar cortex region since no increased microglia proliferative activity is detected in the cerebellar cortex at P10 and P14 (Fig. 5E).

NPC1 deficiency affects microglia differentiation and ramification
In the early days of cerebellar postnatal development, microglial precursors in the WMR were distinctly recognized by their round or ameboid shape ( Fig. 2A-B). It was also evident that as these cells migrate to the cerebellar cortex, where PC dendrites and synaptic connections are developing, they began to differentiate, ramify and extend their processes through the ML where neuronal synaptic connections are found ( Fig. 3C). To determine if NPC1 deficiency alters microglia differentiation, a quantitative analysis of IBA1 + cells morphology was performed in P10 and P14 WT and Npc1 nmf164 mice, using the "Filament Tracer" tool (Imaris). Differences in microglia volume were not detected between WT and Npc1 nmf164 mice at P10 ( Fig.   6A-B), however, at this stage, the microglia total length and the number of terminal points were significantly reduced in Npc1 nmf164 mice ( Fig. 6C-D). At P14, the microglia volume, total length, and terminal points were significantly lower in Npc1 nmf164 mice when compared to WT mice ( Fig. 6A and E-G). These results show that microglia in Npc1 nmf164 mice are less ramified and have shorter processes, suggesting that NPC1 deficiency impairs microglia differentiation and ramification during postnatal development.

Phagocytic activity is increased in developing Npc1 nmf164 microglia
Microglia play an important role in the clearance of apoptotic cells during development (Mosser et al., 2017). While analyzing microglia morphology in P14 postnatal mice, we noticed an abundant number of maturing microglia in the ML containing phagocytic cups, especially in Npc1 nmf164 mice. Phagocytic cups are cupshaped endocytic vacuolar structures in ramified microglia that are formed by the ingestion of particles or cells during phagocytosis (Swanson, 2008). The number of microglia with phagocytic cups was significantly higher in Npc1 nmf164 mice when compared to WT mice ( Fig. 7A-C). We also found that microglia with at least two phagocytic cups and phagocytic cups per image area, were more abundant in the ML of Npc1 nmf164 mice (Fig. 7B, D-E). Interestingly, the number of phagocytic cups containing pyknotic bodies in the ML was higher in Npc1 nmf164 mice than in WT mice, suggesting that NPC1 deficiency increases microglial phagocytic activity in the developing cerebellum. Immunostaining of microglia with the CD68 antibody, a phagosome marker, showed that P14 WT and Npc1 nmf164 microglia were actively phagocytosing at this developmental stage, since CD68 + phagosomes were abundant in microglia from both mouse strains. Markedly, WT IBA1 + microglia at P14 had many small CD68 + phagosomes distributed through the cell body and processes ( Fig. 7G), while in the Npc1 nmf164 microglia the majority of the CD68 + phagosomes were accumulated in the cell body (Fig. 7G). Quantitative analysis showed that the mean volume of CD68 + phagosomes per microglia at P14 was larger in Npc1 nmf164 mice than in WT mice ( Fig. 7G-H), however, no differences were found in the total volume of CD68 between WT and Npc1 nmf164 microglia at this stage (Fig. 7I). Since CLEC7A expression is reactivated specifically in actively phagocytic diseaseassociated microglia (DAM) (Krasemann et al., 2017), to test if P14 phagocytic cells were similar to DAM, P14 and P60 (NPC neurodegeneration stage) WT and Npc1 nmf164 cerebella were immunostained with CLEC7A. We found that CLEC7A + microglia only reappear in the ML of Npc1 nmf164 mice during neurodegeneration at P60 (Fig. S2), and not at P14. Our results suggest that postnatal changes in NPC microglia are developmental alterations caused by NPC1 deficiency and not as an immunological response. These results indicate that NPC1 deficiency alters the phagocytic activity and the distribution of phagosomes in the developing cerebellar microglia.

Development • Accepted manuscript
The role of microglia during this phase of CF synaptic refinement is not completely understood, however recent studies have shown that CF synapse elimination is impaired in mouse cerebella depleted of microglial cells (Kana et al., 2019;Nakayama et al., 2018) suggesting a key role of microglia in CF synapse elimination.
When we examined P14 cerebella from WT and Npc1 nmf164 mice, we found that the volume of CF VGLUT2 + presynaptic inputs in the proximal region of CALB + PC dendrites was significantly reduced in Npc1 nmf164 mice when compared to WT mice ( Fig. 8A-B), as previously reported by others (Caporali et al., 2016). However, we also noticed differences in the distribution of VGLUT2 + inputs between WT and Npc1 nmf164 mice ( Fig. 8A-B). In fact, a higher percentage of CALB + PC somas in Npc1 nmf164 mice contained VGLUT2 + inputs, and in higher numbers (VGLUT2 puncta/soma) than in WT mice ( Fig. 8C-E). Interestingly, significantly more VGLUT2 + inputs in the proximal region of CALB + PC dendrites of P14 WT mice were contacted by IBA1 + microglia than in the Npc1 nmf164 mice (Fig. 8F-H). However, a significantly larger percentage of CALB + PC somas in Npc1 nmf164 mice were contacted by IBA1 + microglia, suggesting a possible link between the excess of CF synaptic inputs in PC somas and the increased interaction of microglia with this region of the PC.
Since previous studies in other regions of the brain have shown that microglia actively engulf and phagocytose presynaptic terminals during developmental pruning (Gunner et al., 2019;Schafer et al., 2012;Tremblay et al., 2010), and P14 cerebellar microglia were evidently phagocytic as they contained high levels of CD68 + phagosomes (Fig. 7G), we analyzed the interaction of individual microglial cells with VGLUT2 + presynaptic inputs. To assess whether IBA1 + microglia are contacting or engulfing VGLUT2 + inputs in WT and Npc1 nmf164 mice, confocal microscopy and 3D surface rendering analysis (Imaris) were used in P14 cerebella. At this stage of Development • Accepted manuscript postnatal development (P14, late phase of CF synapse elimination), WT microglia were actively contacting and engulfing VGLUT2 + inputs in the ML (Fig. 9A).
Quantitative analysis of the total volume of VGLUT2 puncta per microglia at the PCL and ML indicated that Npc1 nmf164 microglia contacted or engulfed significantly more VGLUT2 + inputs than WT microglia at P14 (Fig. 9A-B). By examining the Z-stack sequence images of the microglial cell shown in figure 8A, the interactions of the IBA1 + cell processes with VGLUT2 + inputs innervating CALB + PC dendrites can be observed in Fig. 9C. Some VGLUT2 + inputs were completely engulfed (arrows) by the IBA1 + cell processes, while others were only contacted (Fig. 9C), demonstrating that CF presynaptic inputs are contacted or engulfed by microglia during the late phase of CF refinement. In contrast, Z-stack imaging sequence of the Npc1 nmf164 microglial cell presented in figure 8A clearly shows the interaction of the IBA1 + cell with CALB + PC soma while contacting or engulfing VGLUT2 + inputs that were found abundantly in this region of the PC in P14 Npc1 nmf164 mice (Fig. 9C). These results suggest that NPC1 deficiency not only impairs CF synapse formation, but that it also alters the elimination and translocation of CF synapses in addition to the normal interaction and synaptic pruning function of microglia during the postnatal developmental refinement of CF synapses. Overall, our results show severe impairments in cerebellar microglia and synaptic development that precede and may contribute to early behavioral deficits and neurodegeneration in NPC.

Discussion
In this study, we demonstrate that deficiency of NPC1 affects the postnatal development and function of cerebellar microglia, contributing to profound defects in developmental synaptic pruning and connectivity in the cerebellum. Specifically, we found that lack of NPC1 in mice reduced radial migration, increased proliferation and impaired differentiation of microglial precursors during the first two postnatal weeks.
Increased engulfment of pyknotic bodies and CF presynaptic elements was characteristic of Npc1 nmf164 differentiating microglia at two weeks of age. These Similarly, in the human NPC disease, where the classic presentation of the disease is often found between middle to late childhood, early neurological symptoms associated with cerebellar dysfunction, such as clumsiness, gait disturbances, and eventually ataxia, are observed before the manifestation of other neurological symptoms (Patterson, 1993). These findings suggest that deficiency of NPC1 causes developmental disturbances in the cerebellum that precede neurodegeneration. we hypothesized that NPC1 deficiency severely affects the postnatal development of cerebellar microglia in Npc1 nmf164 mice. Indeed, our data demonstrated that early radial migration of microglial precursors was reduced or delayed in Npc1 nmf164 mice since fewer microglial precursors were found at the IGL in P4 mice. NPC1 deficiency has been previously implicated in the reduced in vitro migration and invasion of CHO and fibroblast cells from NPC patients implicating dysfunctional recruitment and function of integrins in focal adhesion during cell migration (Hoque et al., 2015). It is possible that the intrinsic ability of microglial precursors to migrate is affected by the lack of NPC1 and the lysosomal accumulation of cholesterol.
Another important finding in this study was the increased density of microglia in the developing WMR and cerebellar cortex regions of Npc1 nmf164 mice. In the normal brain, microglia are highly proliferative during the first two postnatal weeks, particularly in the developing CbM and WMR (Li et al., 2019;Nikodemova et al., 2015). A recent study demonstrated that the density of a subset of microglial precursors named PAM peaks at P7 exclusively in the cerebellar WMR (Li et al., 2019). In our study, we found higher proliferative activity in microglial precursors at P4 in the CbM, followed by a significantly increased number of microglia in the cerebellar WMR and in the PCL/ML of P10 Npc1 nmf164 mice. Furthermore, the number of CLEC7A + PAM in the WMR was also increased in Npc1 nmf164 mice suggesting that NPC1 deficiency amplifies the proliferative activity of microglial precursors during highly proliferative stages. An increased number of microglia was still found at P14 and in post-weaning Npc1 nmf164 mice (Kavetsky et al., 2019), indicating that the active proliferation of microglial precursors in the WMR leads to a higher number of these cells in the cerebellar cortex. A few CLEC7A + differentiating microglia were observed in Npc1 nmf164 mice at P10, but these cells were no longer seen at P14 indicating a possible failure of these cells to rapidly downregulate  (Zhao et al., 2018). It is highly probable that NPC1 deficiency causes the pathological changes in developmental microglia through the overactivation of the mTOR pathway, since increased proliferation, impaired differentiation and increased phagocytic activity were hallmarks of postnatal Npc1 nmf164 microglia. Further studies are warranted to determine the role of the mTOR signaling pathway in NPC microglia pathology.
Microglial cells play an important role in the clearance of apoptotic cells during neuronal developmental death (Ashwell, 1990;Mosser et al., 2017). However, it has been also demonstrated, that microglia can induce apoptosis in the neurons they Development • Accepted manuscript phagocytose (Mosser et al., 2017). In this study, an abundant number of maturing microglia in the ML containing phagocytic cups in both WT and Npc1 nmf164 mice were found at the end of the second postnatal week. It was also evident that the number of phagocytic cups and phagocytic cups containing pyknotic bodies was significantly higher in Npc1 nmf164 mice than in WT mice. A high content of phagosomes in P14 microglia at the ML confirmed that at this stage of postnatal development microglial cells were engaged in phagocytic activity. Previous work in the developing rat cerebellum found that the density of phagocytic cups peak around P17 (Perez-Pouchoulen et al., 2015), supporting our findings that microglia is highly phagocytic by the end of the second postnatal week. It is presumed that pyknotic bodies observed at the ML are apoptotic granule precursor cells that were migrating from the ECL into the IGL during postnatal development (Wood et al., 1993).
Interestingly, a reduced number of cerebellar granule cells and reductions in cerebellar lobule size at the end of postnatal development have been found in Npc1 -/mice (Nusca et al., 2014). It is possible that the increased number of phagocytic cups and the engulfed pyknotic bodies in Npc1 nmf164 mice are caused by the increased number of noninflammatory microglia in the developing mutant cerebellum, which could also increase the developmental apoptotic death of cells at the ML. Indeed, an increased number of apoptotic cells was found in mice with elevated microglial phagocytic activity due to the constitutive activation of the mTOR pathway in noninflammatory microglia (Zhao et al., 2018), suggesting that phagocytic microglia can induce and increase developmental neuronal apoptosis. Here we found that VGLUT2 + synaptic inputs from CFs were significantly reduced in the ML of Npc1 nmf164 mice at P14, suggesting that NPC1 deficiency affects CF synapse formation. Our results also indicate that translocation of CF synaptic inputs from the PC soma to the proximal region of PCs dendrites was impaired since a higher number of PC somas contained VGLUT2 + and a greater number of VGLUT2 + puncta per PC soma were found in Npc1 nmf164 mice. A previous study found that not only the glutamatergic CF synaptic inputs were reduced in Npc1 nmf164 mice, but also the GABAergic (basket/stellate cells) inputs, indicating that deficiency of the NPC1 protein broadly impairs synaptic connectivity in the cerebellum (Caporali et al., 2016). These synaptic defects were also associated with developmental deficits in motor skill acquisition in the Npc1 nmf164 mouse.
Importantly, previous studies have demonstrated that microglia have a role in developmental activity-dependent synaptic pruning in the brain (Gunner et al., 2019;Schafer et al., 2012;Tremblay et al., 2010). In fact, microglia engulf and remove intact presynaptic elements during the process of developmental synaptic pruning (Gunner et al., 2019;Schafer et al., 2012;Tremblay et al., 2010). The role of microglia in developmental CF synapse refinement is not completely understood.
However, recent studies have shown that genetic or pharmacological depletion of microglia in the cerebellum impairs the early and late stages of CF synapse elimination during postnatal development leading to behavioral and motor deficits Development • Accepted manuscript (Kana et al., 2019;Nakayama et al., 2018). Furthermore, it is thought that microglia facilitate developmental CF synapse elimination by promoting GABAergic inhibition of PCs (Nakayama et al., 2018). Here, we aimed to determine if cerebellar microglia engulf CF presynaptic inputs at P14 (late-phase of CF synapse elimination) and if NPC1 deficiency alters this microglial pruning function. In fact, we found that at P14, microglia were contacting and engulfing VGLUT2 + inputs in the ML of WT mice.
These results are in accordance with the abundant density of CD68 + phagosomes observed in P14 microglia, indicating that microglia are highly phagocytic in the cerebellum at this postnatal age. Interestingly, at this age, the increased density of somatic VGLUT2 + in Npc1 nmf164 PCs coincided with a higher percentage of PC somas contacted by microglia. Furthermore, P14 microglia contacted and engulfed more VGLUT2 + inputs in Npc1 nmf164 mice than in WT mice. It is possible that the reduced elimination and translocation of VGLUT2 + inputs in Npc1 nmf164 PC somas could be the consequence of decreased GABAergic stimulation to PCs (Caporali et al., 2016), which is also modulated by microglia (Nakayama et al., 2018). Also, it is thought that microglia preferentially engulf and remove presynaptic inputs with decreased activity (Gunner et al., 2019;Schafer et al., 2012;Tremblay et al., 2010), which could explain why a higher number of VGLUT2 + inputs are contacted or engulfed by microglia in Npc1 nmf164 mice. Disrupted presynaptic terminals in NPC can predispose neurons to early neurodegeneration, as demonstrated in a mouse model of the lysosomal storage disease mucopolysaccharidosis type IIIA, where restoration of presynaptic function delayed neurodegeneration (Sambri et al., 2017). Current work in our laboratory is investigating if this phagocytic activity of NPC microglia affects other synaptic refinement and remodeling programs in PCs. Overall, our data demonstrate that deficiency of NPC1 affects microglia and synapse development during the postnatal development of the cerebellum, leading to behavioral deficits and predisposing PCs to neurodegeneration.

Animals
All experiments involving mice were conducted in accordance to policies and procedures described in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Animal Care and Use Committees at the Rowan University School of Osteopathic Medicine. The C57BL/6J-Npc1 nmf164 /J mouse strain (Jax stock number 004817) was provided by Dr.
Robert Burgess at The Jackson Laboratory. Npc1 nmf164 heterozygous mice were bred and housed in a 12/12-hour light/dark cycle to generate both WT and Npc1 nmf164 homozygous mutant mice. Both males and females were used in this study, at a ratio of 2:2 when 4 mice were used.

Mouse Perfusion and Tissue Preparation
Mice were euthanized with CO2 and transcardially perfused with 1X PBS followed by 4% paraformaldehyde. After perfusion, mice were decapitated and their brains were carefully dissected and fixed by immersion in 4% paraformaldehyde overnight. After fixation, brains were rinsed in 1X PBS, immersed in 30% sucrose/PBS solution overnight at 4°C, frozen in OCT, and cryosectioned at 25μm or 50μm (floating sections).

Immunohistochemistry
For immunostaining, brain sections in slides (25μm) or as floating sections (50μm) were rinsed once in 1X PBT (PBS + 1% Triton 100X) and incubated in primary antibodies diluted with 1X PBT + 20% normal donkey serum for overnight at 4°C. After incubation with primary antibodies, sections were rinsed three times with 1X PBT for 10 min and incubated for two hours in the corresponding secondary antibodies (1:800, Jackson-ImmunoResearch or Invitrogen). Tissue was then

Microscopy image analysis
To keep consistency between samples, imaging and quantitative analyses to determine changes in the number of IBA1 + cells and IBA1 + /KI-67 + cells were performed in the first four anterior cerebellar lobules (I-IV). For quantification of IBA1 + and IBA1 + /KI67 + cells in the developing cerebellum, four images (1 per lobule) were taken from two cerebellar cryosections for each mouse (8 images per mouse) with an inverted Leica DMi8 fluorescent microscope. For the WMR, one image per section (two sections) were taken using the Kyence microscope. The imaged regions were randomly selected and investigators were blinded to the genotype. Once the images were taken, a box of 250 X 350 pixels (cerebellar cortex) or 350 X 450 pixels (CbM) was used to crop the images (1-2 boxes per image), so that the area used for the cell counting was consistent between images/animals, and included IGL, PCL/ML, EGL and PS in P4 mice, PCL/ML, EGL and PS in P10 mice, and PCL/ML, EGL and PS in P14 mice. The cropped images were manually counted using the cell counter plugin from the ImageJ (1.47 d) software. For quantification of Lectin + IGL capillaries total length, a region of 400 X 300 pixels was cropped and the Simple Neurite Tracer from ImageJ was used to trace the capillaries and obtain the length measure of every capillary in the image. For quantification of CLEC7A/IBA1 area and MBP intensity, two images per section were taken using the Kyence microscope.
The measure of CLEC7A/IBA1 and MBP immunostained area was selected by threshold and measured by the Analyze plugin of ImageJ. Investigators were blind to the genotype of the tissue while counting the cells or immunostained areas.
For 3D image reconstructions and analyses, three sagittal 50μm cerebellar sections were immunostained by free floating immunohistochemistry. All the images analyzed by the Bitplane Imaris software, were acquired using a Nikon A1R Confocal System equipped with Live Cell 6 Laser Line and Resonant Dual Scanner.
Confocal image stacks were acquired using a 40X objective lens with a 1μm interval through a 50μm z-depth of the tissue. Three confocal images per mouse were taken from the first three lobes (1 per lobe), in the CbM (P4), in the WMR (P10) and in the cerebellar cortex (P10 and P14). To quantify microglial precursors in the WMR of P10 mice, a box of 500 X 500μm was used and Imaris surface rendering tool was used to calculate the volume of IBA1 + cells and colocalization of IBA1 + and KI67 + cells inside the box. The quantification of microglia with phagocytic cups and the number of phagocytic cups were quantified manually in 40X confocal images of the Development • Accepted manuscript ML in P14 cerebella using the cell counter plugin from ImageJ. Two to three images per mouse (n=4) were used for the quantifications in confocal images.
Quantitative analysis of 3D microglia morphology was performed using the Surface rendering tool for cell volume and the Filament Tracer for processes volume and ramification, both tools are part of the Bitplane Imaris software. Confocal zstack images of ~50μm were taken and twenty IBA1 + (5-6 per mouse, n=4 mice) were segregated using 3D surface rendering to be used for the Filament Tracer tool that determines processes length, volume and ramification. The 3D surface rendering was also used to segregate IBA1 + microglia and quantified CD68 + phagosomes inside microglia, or VGLUT2 + synaptic terminals contacted or engulfed by microglia, by using the "Mask all" tool which creates a new channel of the immunostained areas that are inside the created surface (in this case IBA1 surface) and clearing all the fluorescence that is not found overlapping/contacting the rendering surface. The sum of the CD68 or VGLUT2 volume contacted or inside the IBA1 surface was calculated and provided by the software and used for the data analysis presented here. The quantifications of VGLUT2 volume in the ML of P14 mice was performed by cropping the ML region (300μm height X 400μm wide) in 40X confocal images and creating a 3D surface rendering that was used to obtain the sum of the volume of all the VGLUT2 + inputs inside the cropped image. To quantify the volume of VGLUT2 + inputs contacted or engulfed by microglia in the ML, the "Mask all" tool, which creates a new channel of the IBA1 immunostained area that are in contact or inside the created surface (in this case the VGLUT2 surface) was used, then a new surface was created for the IBA1/VGLUT2 overlapping inputs and the calculated volume sum values were collected. The quantification of the percentage of CALB + PCs with VGLUT2 + inputs and the number of VGLUT2 + inputs Development • Accepted manuscript per cell were quantified manually in 1μm Z-sections from 40X confocal images using the cell counter plugin from the ImageJ (1.47 d) software. Two to three images per mouse (n=4) were used for these quantifications.

Quantitative Real-Time Polymerase chain reaction (PCR) array
To measure gene expression changes in mouse innate immune response genes in the cerebellum of WT and Npc1 nmf164 mice Real-Time PCR array TaqMan™ Array Mouse Immune Response. Cerebella from P30 WT (n=4), P30 Npc1 nmf164 (n=4) and P90 Npc1 nmf164 mice (n=3) were collected after mice were perfused with 1X PBS and treated overnight in RNAlater for long-term storage. For RNA extraction, 30mg of cerebellum from each mouse was used and total cellular RNA was extracted and purified from each individual tissue according to the TRIzol™ Plus RNA Purification Kit (ThermoFisher) manufacturer protocol; RNA concentration and purity were determined using the Qubit 2.0 Fluorometer using the RNA quantification kit (Invitrogen). RNA (1 g) was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermofisher). Real-time quantitative PCR was performed using the 96-Well TaqMan™ Array Mouse Immune Response (ThermoFisher) according to the manufacturer protocol and one PCR array plate per mouse was used. Briefly, cDNA samples were diluted appropriately, 540 l cDNA template was added to 540 l of 2X real-time quantitative reaction mixture (TaqMan™ Fast Advanced Master Mix, ThermoFisher), and 10 l of reaction liquid plus cDNA were added to each well of the PCR array, containing gene specific primers. Conditions for the real-time quantitative PCR reaction were as follows: UNG incubation 50°C for 2 min, enzyme activation 95°C for 20 seconds, 40 amplification cycles of denaturing at 95°C for 3 s, annealing/extension at 60°C for 30 s, followed Development • Accepted manuscript by acquisition of fluorescence signal. Data analysis is based on the ΔΔCt method with normalization of raw signal data to housekeeping genes incorporated on the TaqMan™ Array Mouse qPCR plate.

Statistical Analysis
Data were analyzed using GraphPad Prism software. Significance was calculated using unpaired t tests for comparisons between two groups. p-values are provided as stated by GraphPad Prism software and significance was determined with pvalues less than 0.05.  F) The number of IBA1 + /KI67 + cells is significantly higher in the CbM of Npc1 nmf164 mice. G) The percentage of KI67 + microglia is significantly higher in the CbM of Npc1 nmf164 mice. H) IBA1 + and KI67 + cells in the developing cerebellar cortex at P4.
I) The number of IBA1 + /KI67 + cells is significantly lower in the IGL of Npc1 nmf164 mice.