Cerebellar Dysfunction in a Mouse Model of Childhood-Onset Manganese-Induced Dystonia Parkinsonism

Abstract

Humans with pathogenic variants of the manganese (Mn) transporter gene SLC39A14 exhibit highly elevated brain Mn concentrations and childhood-onset dystonia-parkinsonism. Here we show that Slc39a14-knockout (KO) mice, a preclinical model of the disease with elevated Mn concentrations in the CB, express deficits in physiological tremor implicating cerebellar (CB) dysfunction. Imaging of intracellular Mn in Purkinje cells (PCs) using synchrotron-based X-ray fluorescence microscopy confirmed highly elevated Mn concentrations in the PCs of Slc39a14-KO mice. To determine biological pathways altered in the CB of Slc39a14-KO mice relative to wildtype (WT), we performed RNA sequencing and discovered significant upregulation of pathways and genes regulating immune response and cell death. To substantiate these findings, we performed quantitative autoradiography of the neuroinflammation biomarker Translocator Protein 18kDa (TSPO) which was significantly increased in the CB of Slc39a14-KO mice relative to WT. The latter findings were confirmed via immunostaining with the microglial marker Iba-1, revealing widespread microglia activation and clustering in the CB cortex. Immunostaining for cleaved caspase-3 (cCASP3), a marker of apoptosis, showed increased number of PCs with positive immunolabeling for cCASP3 in Slc39a14-KO mice relative to WT. Degeneration of PCs was confirmed by Hematoxylin and Eosin (H&E) staining. Lastly, functional electrophysiological assessment of CB neurocircuitry revealed a marked decrease in firing rates of cerebellar nuclei (CN) neurons and increased variability of PCs simple spikes firing. Collectively, these findings show, for the first time, Mn-induced PCs degeneration and dysfunctional CB circuitry in Slc39a14-KO mice providing additional evidence for the pathological underpinnings of the dystonia-like movements, balance, and gait abnormalities in SLC39A14 mutation carriers.

Introduction:

Childhood-onset manganese-induced dystonia-parkinsonism is a rare and debilitating motor disorder resulting from loss-of-function mutations in the SLC39A14 gene (1-19). Humans with loss-of-function mutations in the SLC39A14 gene exhibit highly elevated systemic and brain manganese (Mn) concentrations resulting in a progressive childhood-onset dystonia-parkinsonism classified as hypermanganesemia with dystonia 2 (HMNDYT2; OMIM#617013) (1, 4-12). SLC39A14 is a Mn influx transporter that was originally hypothesized to be a zinc influx transporter. However, recent evidence shows that SLC39A14 is primarily a manganese (Mn) transporter with some affinity for other divalent cations (1-19). The clinical manifestation of the neurological deficits in affected children include progressive generalized dystonia, bulbar dysfunction and loss of developmental milestones (1, 4-12). Within the first decade of life, mutation carriers develop severe dystonia, spasticity, loss of independent ambulation, and in some cases, parkinsonian features with hypomimia, tremor, and bradykinesia. While dystonia is central to the clinical manifestation of the disease, the underlying pathophysiology and the cellular basis for the behavioral abnormalities in the mutation carriers remains largely unexplored (1, 4-12).

Analysis of the peer-reviewed literature since the first description of SLC39A14 mutation cases by Tuschl and colleagues (1), indicates that mutation carriers exhibit over 20-fold increase in blood Mn concentrations and highly elevated brain Mn levels based on T1-weighted Magnetic Resonance Imaging (MRI) (1, 3-12). Brain volume changes in mutations carriers, based on MRI imaging, is atrophy of the cerebellum (CB) (1, 4-12). Atrophy of the CB folia and dentate nuclei have been noted in other clinical report by Marti-Sanchez and colleagues further supporting the hypothesis of CB involvement in the pathology of the disease (1, 7). Because there has been a lack of pathological studies in humans and limited access to postmortem brain tissue, preclinical animal models have been developed to study the pathophysiology of this devastating pediatric disease (2,13-19).

Our laboratory has previously characterized a Slc39a14 knockout (KO) mouse model of the disease from the perspective of the nigrostriatal dopaminergic system to understand the dystonia and parkinsonian features exhibited by SLC39A14 mutation carriers (2). We (2, 13) and others (14-19) have shown that the Slc39a14-KO mice express locomotor, balance, and gait deficits and express dystonia-like movements and elevated blood and brain Mn levels. The complex behavioral phenotype exhibited by the Slc39a14-KO mice resembles the neurological deficits observed in the human cases of the disease. Because dystonia and parkinsonism are motor disorders thought to originate in the basal ganglia, our initial studies focused on the nigrostriatal dopaminergic system (2, 26). We discovered that in young (60 days of age) and in aging (365 days of age) Slc39a14-KO mice there was a dramatic inhibition of potassium-stimulated dopamine (DA) release in the striatum with no degeneration of dopaminergic neurons in the substantia nigra pars compacta relative to age- and sex-matched wildtype (WT) mice (2, 13).

Human cases of the disease and Slc39a14-KO mice have a very complex behavioral phenotype that is unlikely to be solely explained by the deficits in striatal DA release that we have observed (1-2, 4-19). Growing evidence from basic and clinical research indicates that dystonia may arise from motor network dysfunction that spans both, the basal ganglia and the CB (20-24). Because the CB plays an important role in dystonia, gait, posture, and balance and CB atrophy has been reported in the clinical literature describing a limited number of human cases with the disease (1, 4-12), we examined behaviors mediated by the CB (24, 25) and analyzed the principal CB cortex output cells – Purkinje cells (PCs) from a neuropathological and functional perspectives (27). Our findings describe for the first time PCs degeneration in the CB of Slc39a14-KO mice. This finding provides new evidence that adds to our understanding of the pathophysiology of this devastating disease arising from pathogenic mutations in SLC39A14.

Results:

Tremor analysis:

The spectrum of symptoms found in dystonia-parkinsonism is broad (1, 22, 26). However, a movement abnormality that is frequently associated with parkinsonism and movement disorders is tremor (26). Therefore, to investigate tremor in the Slc39a14-KO mice, we performed tremor analysis in PN60 male and female animals. For this purpose, we used a tremor monitor (San Diego Instruments) and discovered an average decrease in tremor amplitudes across all measured frequencies from 1-64 Hz in Slc39a14-KO PN60 male (WT, n=10; KO, n=11) (F (1, 19) = 10.71; p=0.004) and female (WT, n=5; KO, n=5) (F (1, 8) = 59.25; p<0.0001) mice, when compared to sex and age matched WT controls (Figure 1A and B; Supplementary Figure 6A and B for full spectrum). Statistically significant decrease in tremor amplitudes in males was observed at frequencies of 2-43 Hz, 45 Hz and 50-52 Hz, while females presented with a statistically significant decrease across all frequencies measured (1-64 Hz). We specifically found a significant decrease in tremor amplitudes in frequencies between 6-13 Hz in both male and female Slc39a14-KO mice (Figure 1A-B; black dotted bar above the x-axis). This range of frequencies denotes the range of normal physiological tremor in mice and humans, which is centered at 10 Hz. Reduction in baseline physiological tremor has previously been linked to a constitutive lack of PCs fast GABAergic neurotransmission via conditional knockout of the Vesicular GABA Transporter (VGAT) (24). In the Slc39a14-KO mice, a decrease in physiological tremor and other frequencies implicate broad dysfunction of the CB neurocircuitry.

Figure 1. Tremor analysis in WT and Slc39a14-KO PN60 mice.

A) Tremor frequencies between 1-25Hz in WT and Slc39a14-KO PN60 male mice. Significant decrease in the tremor amplitudes of Slc39a14-KO PN60 male mice was noted at frequencies 2-25Hz (WT n=10; KO n=11) (F (1, 19) = 10.71) (p=0.0040). B) Tremor amplitudes from 1-25Hz in WT and Slc39a14-KO PN60 female mice. Significant decrease in tremor amplitudes of Slc39a14-KO PN60 female mice was noted across the entire spectrum of 1-25Hz. (WT n=5; KO n=5) (F (1, 8) = 59.25) (p<0.0001). Values are represented as means +/− SEM. Frequency band of 6-13Hz, which corresponds to normal physiological tremors, is denoted as a black dotted line. Colored bars represent the range of highest and lowest values. Mixed effects model with FDR adjustment across multiple comparisons was performed using the two-stage step-up method of Benjamini, Krieger and Yekutieli.

Metals Analysis in the Cerebellum:

We had previously characterized metals concentrations in the striatum of Slc39a14-KO and WT mice (2, 13). However, T1-weighted MRI imaging in the human cases of the disease reveals that Mn accumulation is not only localized to basal ganglia structures but is also observed throughout the brain, including the CB (1, 4-12). Furthermore, the SLC39A14 patients present with progressive CB atrophy (1). Therefore, to further characterize the Slc39a14-KO murine model of the human disease and to investigate brain region-dependent patterns of Mn distribution and accumulation, we performed inductively coupled plasma-mass spectrometry (ICP-MS) metals analysis in whole CB from PN60 Slc39a14-KO and WT male and female mice. Consistent with the distribution patterns seen in the human cases of the disease (1, 4-12), our ICP-MS studies revealed a marked increase in Mn concentrations in the CB of male (13-fold increase, p=0.0005, WT, n=6; KO, n=6) and female (8.4-fold increase, p=0.0002, WT, n=6; KO, n=6) Slc39a14-KO PN60 mice, when compared to age and sex matched WT controls (Figure 2A). We also discovered a small but significant increase in CB Fe concentrations (1.2-fold increase, p=0.0497) in males, and a decrease in CB Cu (0.7-fold decrease, p=0.0086) and Zn (0.74-fold decrease, p=0.0131) in females (Figure 2A).

Figure 2. Whole tissue and cellular metals concentrations in the cerebellum of WT and Slc39a14 KO PN60 mice.

A) Whole tissue CB metals concentrations. Mn and Fe levels in WT and Slc39a14-KO PN60 males are markedly elevated: 1,195% (p=0.0005) and 20.6% (p=0.0497) increase, respectively. Female Slc39a14-KO mice also present with elevated CB Mn concentrations: 774% increase )p=0.0002), while Cu and Zn are decreased: 30.48% (p=0.0086) and 26.3% (p=0.0131), respectively, (WT n=6; KO n=6 for males; WT n=6; KO n=6 for females). ICP-MS data were analyzed using Welch’s t-test, values are represented as means +/− SEM. B) Raw XRF spectra acquired at Beamline 9-ID Bionanoprobe, Advanced Proton Source, Argonne National Laboratory. C) Raw XRF spectra acquired at 3-ID HXN, NSLS-II, Brookhaven National Laboratory. Blue and red colors represent Slc39a14-WT and Slc39a14-KO PN60 males, respectively. All marked peaks correspond with K-α emission lines. Mn levels are increased in the Slc39a14-KO when compared to the WT control, while Fe, Cu and Zn remain unchanged. D) Quantification of PCL metals concentrations from APS and BNL spectra. Mn levels are highly elevated in the PCL of Slc39a14-KO mice, compared to WT controls, with Fe, Cu and Zn levels remaining unchanged (WT n=3; KO n=3; 17-fold increase; p=0.0433). XRM data were analyzed using Kruskal-Wallis test, values are represented as means +/− SEM.

Synchrotron-based X-ray Fluorescence Microscopy:

To delineate the spatial and cellular distribution of Mn, Fe, Cu, and Zn in the CB of WT and Slc39a14-KO mice, we performed synchrotron-based X-ray fluorescence microscopy (XFM) at the Advanced Photon Source (APS) of the Argonne National Laboratory (Figure 2B for raw spectra) and National Synchrotron Light Source II of the Brookhaven National Laboratory (BNL) (Figure 2C for raw spectra).

For synchrotron radiation-based XFM imaging, we used 10μm thick sagittal CB sections from PN60 Slc39a14-KO and WT males (29). The samples were imaged at the level of the vermis demonstrating 4 distinct cellular layers – molecular cell layer (MCL), granular cell layer (GCL) and Purkinje cell layer (PCL); along with the corresponding white matter tract (WM). Owing to its unique morphology and localization between the MCL and GCL of the CB, it is possible to identify the PCL using elemental spatial distribution images (WT: Supplementary Figure 1 A-1 and Supplementary Figure 3 A1-4; Slc39a14-KO: Supplementary Figure 1 B-1 and Supplementary Figure 4 A1-4; pixel size ~1.5 μm for tissue level low resolution imaging). Mn distribution in Slc39a14-KO brain appears to be clustered along the PCL (Supplementary Figure 1 B-1 and 4 A-1, Mn). No such clustering is observed along the border of the MCL and GCL in the WT image (Supplementary Figure 1 A-1 and 3 A-1, Mn).

To understand the cellular and intracellular Mn distribution in the CB, XFM elemental images were obtained at cellular (pixel size ~100nm; Supplementary Figure 2 A-B, Supplementary Figure 3, and Supplementary Figure 4-1) and intracellular levels (pixel size ~70 nm; Supplementary Figure 1 A2-A5 and B2-B5) of spatial resolution. In Supplementary Figure 1 A and B, panels 2-5, display ROIs are shown as white squares in low magnification images in the Slc39a14-KO and WT tissue, respectively. PCs localization within the high magnification image is denoted as white dashed outlines. Slc39a14-KO XFM image of Mn (Supplementary Figure 1 B2) demonstrates significantly higher accumulation of Mn in the PCL, while no such accumulations are evident in the WT Mn XFM image (Supplementary Figure 1 A2). To visualize the relative distribution of elements in Slc39a14-KO (Supplementary Figure 2) for Mn/Cu and Fe/Cu, however, no distinct colocalization was observed. In Supplementary Figures 2 A and C areas rich with Cu aggregates (30) (white arrows) surround Mn depositions that are distributed within the PCL. This distribution might indicate that cells with Mn and Cu are two different cell types that are located in close proximity. The punctate appearance of Cu distribution around the PCL (Supplementary Figures 1-2), may arise from Bergmann glia (BG) (28).

XFM imaging was repeated at the Hard X-ray Nanoprobe (HXN) beamline 3-ID, NSLS-II, BNL (73). The whole area averages of all elemental XFM images (Figure 2C) obtained at NSLS-II are reported in Supplementary Tables 4 and 5. When assessing the raw spectral data (Figure 2C; blue line is WT, red line is KO) no significant changes can be observed in Fe, Cu, and Zn in KO compared to WT (Supplementary Tables 4 and 5). Data obtained at two different beamlines (Supplementary Tables 2-5) while varying in absolute elemental concentrations possibly due to differences in the quantification methods or exact areas of CB analyzed, overall show the same order of magnitude change in Mn concentrations. The quantitative analysis of selected cells obtained XFM images (Supplementary Figures 1, 3 and 4) from PN60 Slc39a14-KO and WT males, n=3 per group (n=1 was imaged at the APS facility and n=2 were imaged at the NSLS-II) are reported in Figure 2D. Overall, we observed an average increase of ~17-fold in Mn content within the Purkinje Cellular Layer of Slc39a14-KO CB samples (n=3 for WT and n=3 for KO, p=0.0433 for Mn with no changes in Fe, Cu or Zn in PN60 males, data analyzed using Kruskal-Wallis test). While Mn concentration in the PCL of WT CB is 2.26 μg/g, it is 40.11 μg/g in the Slc39a14-KO mice, or approximately a 17.7-fold increase in the PCL as seen in Figure 2D. These findings are consistent with a previous study using MRI in mice indicating that administration of high doses of Mn selectively increases Mn levels in PCs (31).

Analysis of Differentially Expressed Genes Through RNA Sequencing:

We performed RNA isolation and sequencing to investigate putative changes in the transcriptome of the Slc39a14-KO mice in response to markedly elevated Mn levels in the CB. We isolated RNA from freshly harvested whole CB from WT and Slc39a14-KO PN60 mice. The isolated and purified RNA was sent to Novogene for sequencing and analysis. Gene ontology (GO) analysis revealed a marked upregulation of pathways responsible for mounting an immune response, positive regulation of cytokine production and upregulation of defense responses (Figure 3A). To further investigate the degree of change in differentially expressed genes (DEG), we visualized the RNAseq data as volcano plot (Figure 3B). We specifically discovered an increase in Oasl2, a facilitator of RIPK3 activation, MLKL phosphorylation and necrosome formation; Ifi27, which has been shown to positively modulate activation of various caspases, release of cytochrome C and cell death, and Irf7 which is a well-established regulator of interferon production (Figure 3B) (32-34) (WT, n=3; KO, n=3, in males). For more gene-pathway functions, see Supplementary Table 6. The gene expression data indicate that in the presence of elevated Mn levels, the CB of Slc39a14-KO mice manifest an increase in biological pathways that are tightly linked to cell death and immune response.

Figure 3. RNA sequencing in bulk cerebellar preparations reveals marked upregulation of immune modulating pathways, pro-apoptotic and pro-inflammatory genes.

A) RNA-seq gene ontology dot plot of top 20 upregulated pathways. The size of the circle represents the count numbers for the pathway of interest, the pseudo color coding for the adjusted p-values represents significance of change in a given pathway with red color denoting a p-value closer to 0. Note the marked upregulation pathways responsible for mounting an immune response. B) Volcano plot of upregulated and downregulated differentially expressed genes (u/dDEG). Data points in light blue and light red represent identified DEGs before FDR correction, while dark blue and dark red data points outlined in black represent FDR-corrected DEGs. 1.3 on the y-axis represents the −log10 of p=0.05 for the uncorrected p-values. PN60 WT and Slc39a14-KO males were used for RNA extractions (WT n=3; KO n=3). Log2 Fold Change was set at 1. Data were sequenced and analyzed by NovoGene, GraphPad Prism 10.4.1 was used to visualize the data.

TSPO Autoradiography:

Translocator Protein 18 kDa (TSPO) is a well validated biomarker of neuroinflammation and brain injury and has been extensively used to evaluate neuroinflammation in the brain without a priori knowledge of the affected areas (35-38). Quantitative unbiased TSPO autoradiography was performed in PN60 male and female WT and Slc39a14-KO mice to investigate potential regions undergoing active neuroinflammation and neurodegeneration. TSPO autoradiography using [³H]-DPA-713 showed that specific binding was increased in the CB of Slc39a14-KO animals relative to sex and age matched WT controls (Figure 4A-B). Using the Allen Brain Atlas we further analyzed specific sub-regions of the CB (Figure 4C). Both male and female Slc39a14-KO mice expressed increased TSPO levels in the CB cortex (p=0.0157 – males, p=0.014 – females) and CN (p=0.0038 – males, p=0.0076 – females) (WT, n=6; KO, n=6 for males; WT, n=6; KO, n=5 for females) (Figure 4D) indicative of ongoing glial cell activation and putative neurodegeneration (35-38).

Figure 4. Quantitative TSPO autoradiography in the cerebellum of WT and Slc39a14-KO PN60 male and female mice.

A-B) Representative autoradiograms of [³H]-DPA-713 binding in the CB of PN60 WT and Slc39a14-KO male and female mice. Red-blue color spectrum corresponds to high and low binding, respectively. Blue outline represents the region of the CB cortex used for quantification and analysis; red outline corresponds with CN. C) Representative image of Nissl stain section of the CB obtained from the Allen Brain atlas at the level of pseudo color images seen in panel (A). Blue outline represents the CB cortex; red outlines denote the CN. D) Quantification of autoradiograms demonstrates a marked increase in TSPO levels across CB cortex in the males (p=0.0157) and females (p=0.0140) and CN in males (p=0.0038) and females (p=0.0076) of Slc39a14-KO mice when compared to age- and sex-matched WT controls, (WT n=6; KO n=6 for males; WT n=6; KO n=5 for females). Data were analyzed using Welch’s t-test and are represented as means +/− SEM.

Histological Assessment of Microglial Activation Using Iba-1 Immunohistochemistry:

Based on the TSPO autoradiography results indicative of neuroinflammation, we performed immunohistochemistry using the microglia marker Iba-1 (39) in sagittal brain sections of PN60 WT and Slc39a14-KO male and female mice (WT, n=3; KO, n=6 in males; WT, n=6; KO, n=4 in females) (Figure 5A-H). Initial assessment revealed lack of microglial clustering and activation in the forebrain of PN60 Slc39a14-KO animals when compared to WT controls. However, several regions of the CB presented with an increase in microglia number in a region and nuclei specific manner. In males, both CB cortex and CN had increased microglia density and clustering, represented as area coverage, while in the females only the CB cortex expressed an increase in microglial area coverage. In contrast, the microglia within the CB of WT animals were distributed homogenously in both sexes. To evaluate percent area covered by the Iba-1+ cells, we performed signal/area analysis using FIJI/ImageJ software. We quantified Iba-1+ percent area coverage in the deep cortical regions (DCR) of the CB in the Slc39a14-KO mice against matched WT controls (Figure 5C-D). To that end, we detected an average increase in microglial area coverage in the Slc39a14-KO male and female mice, with males having a marked and statistically significant increase across lobules 4&5 (p=0.0460), 6 (p=0.0456), 7 (p=0.0465), 9 (p=0.0064), 10 (p=0.0266) and CN (p=0.0323) (WT, n=3; KO, n=3); while females presented with an increase in lobules 4&5 (p=0.0181), 6 (p=0.0022), 9 (p=0.0164) and 10 (p=0.0007) (WT, n=6; KO, n=4) (Figure 5C-D). While percent area coverage of DCR of the CB by microglia provides a picture of pathology in the regions of interest, it does not directly demonstrate microglial activation and shift to a phagocytic phenotype. To further investigate the degree of microglial activation we employed a novel machine learning-based approach of identifying, segmenting and analyzing microglial morphology following Iba-1 DAB-IHC (Figure 5E-H). Micrograph representations of WT and Slc39a14-KO microglia can be seen in Figure 5E and F, respectively. Red masking represents microglia captured in the CB of a WT mouse, while blue masking represents microglia in the CB of an Slc39a14-KO mouse. White dots correspond to the center of the microglial soma. Scale bar is 15 μm. Once identified and segmented, the perimeter of each microglia was calculated using the circularity and area metrics obtained from IMARIS software. Perimeter quantification results for the male and female animals can be found in Figure 5G-H, respectively. The male Slc39a14-KO animals presented with a significantly decreased microglial perimeter, when compared to the WT controls across every lobule analyzed (Figure 5G). A similar pattern of microglial activation in the CB was observed in female mice, however, the degree of significance across the lobules was smaller than that found in the males, with lobules 3-5 and 8-9 presenting with significant reduction in cell perimeter (WT, n=3; KO, n=3 in males; WT, n=4; KO, n=4 in females; (F (15, 32) = 13.18 in males and F (15, 48) = 2.34 in females); every data point represents an average of 10 cells). Overall, the microglial quantification data represent the presence of a pro-inflammatory, phagocytic phenotype as a response to a triggering event, such as neuroinflammation or neurodegeneration as evidenced by TSPO autoradiography.

Figure 5. Histological assessment of microglial reactivity in the cerebellum of WT and Slc39a14-KO PN60 male and female mice.

A-B) Photomicrographs of the CB at 2x and 20x magnifications reveals widespread microglial activation and clustering in the DCR of the CB and CN of male Slc39a14-KO animals when compared to sex-matched WT controls. C-D) Quantification of DCR %-area covered by Iba-1 positive cells within the CB of WT and Slc39a14-KO male and female mice reveals a marked increase in microglial populations across numerous DCR of CB lobules and CN. ROIs were selected in individual lobules of the CB of Slc39a14-KO animals and WT controls. C) Quantification of % area covered by Iba-1 positive microglia in the DCR of male WT and Slc39a14-KO mice. Lobules 4-10 of Slc39a14-KO males present with a marked increase in microglial surface area coverage, clustering and activation, along with the CN. (WT n=3; KO n=3) D) Quantification of % area covered by Iba-1 positive microglia in the DCR of female WT and Slc39a14-KO mice. Lobules 4-6, and 9-10 present with increased microglial activation. No change was observed in the CN at this level of the CB. (WT n=6; KO n=4) Data were analyzed using Welch’s t-test and are represented as mean +/− SEM. E-F) Photomicrographs and IMARIS machine learning based surfaces masks of WT and Slc39a14-KO microglia morphology in male mice (Panels E and F, respectively). White dots represent the center of microglial soma. Scale bar is 15μm. G-H) Quantification of microglial perimeter as computed from circularity and surface area output obtained from machine learning-based detection and segmentation in IMARIS. IMARIS quantification revealed a significant decrease in microglial perimeter in the Slc39a14-KO males when compared to sex and age-matched WT controls in the males (Panel G) and in the females (Panel H). (WT n=3; KO n=3 in males; WT n=4; KO n=4 in females). Data were analyzed using one-way ANOVA (F (15, 32) = 13.18 in males; and F (15, 48) = 2.34 in females). Each point represents an average of 10 microglia per lobule per animal.

Expression of Cleaved Caspase-3 in the Cerebellum:

Based on the TSPO autoradiography and microglia immunostaining results as well as the accumulation of Mn in the PCL, we began to investigate the possibility of PCs degeneration. Cleaved caspase-3 (cCASP3) expression is canonically central to apoptosis (40-41). Therefore, we performed quantitative immunohistochemical assessment of cCASP3 expression in the CB of PN60 WT and Slc39a14-KO mice. Figure 6A demonstrates a low magnification image of CB lobules 4-5 with low levels of cCASP3 expression in the CB of a WT mouse. Figure 6B demonstrates a broad increase in the number of PCs positive for cCASP3 in the CB of Slc39a14-KO mouse when compared to sex and age matched WT controls. Note the distinct absence of cCASP3 staining in the PCL of the CB in the WT animal in Figure 6A, demarcated by a white arrow. The CB of Slc39a14-KO animal has a diametrically opposite staining phenotype, with cCASP3 positive PCs (see white arrow in Figure 6B) and PCs dendritic processes (red arrow in Figure 6B). Quantification of cCASP3 expression in the PCL reveals that the Slc39a14-KO mice express a marked increase in the number of cCASP3 immunoreactive PCs that was lobule specific. In the Slc39a14-KO male animals, the number of cCASP3 positive cells per mm of PCL is significantly increased across lobules 3 through 6, as well as lobule 8 (WT, n=4; KO, n=5; F (13, 49) = 9.237; p=0.0088 to p=0.001). The female mice present with a similar pattern of cCASP3 staining in the PCs of the KO animals, with significant increase in cCASP3 cell numbers observed in lobules 2-6 (WT, n=5; KO, n=5; F (13, 56) = 7.444; p<0.0014 to p<0.0001). Together with the Iba-1 immunostaining and the marked increase in microglial activation and clustering in the CB, the cCASP3 data provides further evidence of an ongoing neurodegenerative processes in the PC layer in the CB of Slc39a14-KO mice.

Figure 6. Histological assessment of neurodegenerative changes in the cerebellar cortex of Slc39a14-KO PN60 male and female mice.

A-B) Photomicrographs of cCASP3 immunofluorescence at 2x and 60x magnification in the male CB of WT and Slc39a14-KO PN60 mice reveals marked and widespread upregulation of cCASP3 expression across the CB, with PCL immediately apparent in the DCR of the CB of male Slc39a14-KO animal. Note the nearly complete lack of cCASP3 immunoreactivity in the low magnification image of the sex-matched WT animal (Panel A). In contrast, the staining intensity and specificity to PCL increases drastically in panel B, corresponding to Slc39a14-KO. Furthermore, as denoted by a white arrow in PCL in high magnification sub-panel A, the PCs are not immunoreactive in the WT animal, while in the Slc39a14-KO panel B, the PCs have highly pronounced levels of cCASP3 in the soma (white arrow) and dendritic projections within the MCL (red arrow). C) Quantification of cCASP3 positive cells in the PC layer of male WT and Slc39a14-KO mice. Lobules 2-6 and 8 were found to have marked increase in cCASP3 levels in Slc39a14-KO mice when compared to WT controls (WT n=4; KO n=5). D) Quantification of cCASP3 positive cells in the PC layer of female WT and Slc39a14-KO mice. Lobules 2-6 were found to have marked increase in cCASP3 levels in Slc39a14-KO mice when compared to WT controls (WT n=5; KO n=5). Data were analyzed using one-way ANOVA with Bonferroni correction for multiple comparisons (F (13, 49) = 9.237 for males; F (13, 56) = 7.444 for females) Data are represented as mean +/− SEM. E) Photomicrographs of H&E staining in FFPE section of Slc39a14-WT CB. White outline in the lower magnification image represents the ROI used for the high magnification photomicrograph. Note the continuous layer of PCs with distinct nuclear hematoxylin staining. F) Photomicrograph of H&E staining in FFPE section of male Slc39a14-KO CB. Compared to the sex-matched WT control, the KO animal CB presents with shrunken and eosinophilic PCs that are pyknotic, indicative of ongoing neurodegeneration.

Hematoxylin and Eosin Staining in the Cerebellum:

To further supplement our findings of cCASP3 immunoreactivity in the PCL of Slc39a14-KO mice, we performed H&E staining in formalin fixed and paraffin embedded (FFPE) sections of CB from PN60 WT and Slc39a14-KO mice. Figure 6E depicts images at different magnifications of the PCL from a PN60 WT male mouse CB. Note the healthy appearance of PCs organized in a uniform layer with distinct hematoxylin staining in the nucleus and pear-shaped soma. In contrast, the H&E stain in the region-matched section of CB from a Slc39a14-KO male seen in Figure 6F reveals presence of eosinophilic PCs with shrunken soma and visible signs of pyknosis (supplementary images from the DCR of lobules 3-6 can be found in Supplementary Figure 5). Taken together with the cCASP3 immunostaining data, H&E-based histology reveals a broad phenotype of neurodegeneration of PCs.

In vivo electrophysiology:

Abnormal spike activity of PCs and cerebellar nuclei (CN) cells is indicative of ongoing disordered movement including ataxic discoordination (42), dystonic posturing (43), and alterations in baseline tremor (24). Aberrations in PC and CB nuclei neuron spike activity have been observed with degeneration of the CB circuit (44, 45). Therefore, we examined the firing properties of PCs and their predominant downstream partners, CN neurons, in awake head-fixed young adult Slc39a14-KO and WT mice.

We first recorded from PCs (Figure 7A-C) and observed no changes in mean firing rate, coefficient of variance (CV) – a measurement of irregularity of the firing rate over the entire analysis window, or CV2 (46) – a measurement of irregularity of spike timing from one spike to the next. Both PC simple spikes and complex spikes had no significant difference in the mean of these measurements across the population of cells recorded (Figure 7D-I). However, signs of degeneration were more prominent in some areas of the CB than others (Figures 5 and 6). Additionally, previous work has shown that mice with various CB pathologies can have a large proportion of cells recorded with healthy firing properties despite the presence of motor phenotypes (47) and PCs contain subpopulations with varying susceptibility to degeneration and cell death (48). Therefore, we tested whether there was a difference in variability in our selected spike measurements to determine whether spike properties may be dysregulated within the population of PCs despite the maintenance of a healthy population mean. We found that all PC simple spike firing features – firing rate, CV, and CV2 – had significantly increased population variability in Slc39a14-KO mice (Figure 7D-F). Complex spikes did not exhibit this change in population variability (Figure 7G-I). See Supplementary Table 7 for detailed F-statistics. Therefore, while complex spike activity is unchanged in Slc39a14-KO mice, there is a greater degree of variability in simple spike activity of PCs from Slc39a14-KO mice compared to WT mice.

Figure 7. Electrophysiological analysis of Purkinje cell activity in awake adult Slc39a14-KO mice.

A) Schematic of extracellular recordings of cerebellar Purkinje cells in adult mice. B-C) Representative 1-second-long Purkinje cell raw spike traces from WT (B) and KO (C) mice. Open arrowhead indicates a complex spike. Scale = 100 ms. D-I) Analysis of Purkinje cell spike activity from adult PN59-161 mice. WT N = 12 and n = 27 (female N = 6 and n = 13), KO N = 10 and n = 22 (female N = 7 and n = 15). T-test and F-test p values are listed above each comparison. Asterisk indicates adjusted p value ≤ 0.05. D-F) Purkinje cell simple spike firing rate (D), CV (E), and CV2 (F) by genotype (above) and sex+genotype (below). WT mean ± SEM is 69.80 ± 2.709 (D), 0.4477 ± 0.01633 (E), and 0.4172 ± 0.01258 (F), respectively. KO mean ± SEM is 76.39 ± 4.987 (D), 0.5002 ± 0.02985 (E), and 0.4652 ± 0.02277 (F), respectively. G-I) Purkinje cell complex spike firing rate (G), CV (H), and CV2 (I) by genotype (above) and sex+genotype (below). WT mean ± SEM is 1.302 ± 0.05222 (G), 0.7575 ± 0.02505 (H), and 0.8807 ± 0.02202 (I), respectively. KO mean ± SEM is 1.342 ± 0.08266 (G), 0.7476 ± 0.02116 (H), and 0.8640 ± 0.01806 (I), respectively.

We then measured spike properties in CN neurons (Figure 1A-C) since nuclei neurons are the main recipient of PC innervation and represent the predominant output from the CB. We found that the mean firing rate was significantly reduced in Slc39a14-KO mice (Figure 1D). The regularity of spike patterning was unchanged both over the entire analyzed period and spike-to-spike (Figure 1E-F). While CN neurons integrate inputs from many PCs (for a dedicated investigation of PC-to-nuclei neuron convergence, see (49)), the projection patterns of PCs are non-random (50-52), therefore we tested whether nuclei cells had similar population variability as was found in PCs. We found that nuclei neuron CV in the CB of Slc39a14-KO mice had increased population variability compared to those from WT mice (Figure 1E), while population variability was similar for firing rate and CV2 (Figure 1D&F). See Supplementary Table 7 for detailed F-statistics. Therefore, output from the CB is abnormally slow in Slc39a14-KO mice and across the CN neuron population there is greater variability in spike patterning exiting the CB to the rest of the motor circuit.

Discussion:

This body of work characterizes for the first time the neuropathological changes in the CB of Slc39a14-KO mice, a murine model of childhood-onset dystonia parkinsonism resulting from loss-of-function mutation of the Mn transporter gene SLC39A14. From a behavioral perspective, we have previously described a complex set of behavioral abnormalities, manifesting as dystonia-like movements and locomotor deficits (2, 13). In the present work, we report the novel finding that Slc39a14-KO young adult male and female mice exhibit an overall decrease in tremor amplitudes. Furthermore, there is a marked and significant decrease in tremor amplitudes at 6-13 Hz, which corresponds to the frequency of normal physiological tremor. Physiological tremor is present in all mammals at baseline and is generated by a confluence of multiple biological and mechanical factors including the underlying electrophysiology of multiple motor areas in the central nervous system, the electrophysiology of muscles, oscillatory mechanics of reflexes and movements of limbs, even an animal’s heartbeat contributes to physiological tremor (25). Decreased physiological tremor has been described in mice in which the vesicular GABA transporter (VGAT) was genetically deleted from PCs, resulting in constitutive silencing of PCs GABAergic neurotransmission and altered caudate nucleus (CN) firing properties (24). Like the Slc39a14-KO animals, the VGAT mutant mice present with uncoordinated, ataxic movements (24). However, unlike in the VGAT deletion model, the Slc39a14-KO animals also present with dystonia-like movements (2, 13, 17). Taken together, the current neuropathological studies begin to elucidate the complex behavioral phenotype seen in the Slc39a14-KO murine model of childhood-onset Mn-induced dystonia-parkinsonism described in individuals with pathogenic mutations in SLC39A14 (1-2, 4-19).

The dystonic movements found in the Slc39a14-KO mice may be explained by several factors: 1) inhibition of DA release in the striatum of the Slc39a14-KO mice (2, 13), 2) Mn induced neuropathological changes in CB cortical neurocircuitry which may not be limited exclusively to PCs, and 3) dysfunction/degeneration in the CN. However, the exact cause is likely a combination of multiple factors that remain to be fully elucidated. To investigate potential neuropathological changes in the context of elevated CB Mn concentrations in the Slc39a14-KO mice, we measured Mn concentrations in the whole CB using ICP-MS and at the PC cellular level using XFM. We found highly elevated CB Mn concentrations in PN60 male and female Slc39a14-KO mice relative to WT. Furthermore, analysis of PCs Mn concentrations using XFM revealed highly elevated Mn concentrations at the cellular level. These findings are consistent with a previous study using Mn-enhanced T1-weighted MRI in which they show a selective accumulation of administered Mn in PCs (31). It is possible that Cu localization seen in the form of puncta depicted in Supplementary Figure 1B and C and Supplementary Figure 2 could be localized within BG (37). BG are a specific subtype of astrocytes and are found exclusively in the PCL of the CB, with approximately 4 BG per PC, their radial projections extend across the molecular layer, while the BG soma surrounds the PC soma (53).

High levels of Mn in the brain have long been known to be neurotoxic (76). To interrogate the effects of elevated Mn levels on gene expression in the CB of Slc39a14-KO mice, we performed RNA sequencing studies using whole CB preparations. We discovered that in the presence of highly elevated Mn levels, the Slc39a14-KO mice present with markedly upregulated biological pathways that are responsible for immune response, host defense and pro-inflammatory processes. Furthermore, we also discovered an increase in genes that have been linked to pro-apoptotic and -necroptotic processes mediated by cytochrome-c release, activation of several caspases and RIPK3 fibrillation (32-34). See Supplementary Table 6 for further breakdown of genes and their functions. To further expand on our RNAseq findings demonstrating increased genes associated with the immune response and pro-inflammatory processes, we performed unbiased quantitative TSPO autoradiography in the CB of WT and Slc39a14-KO mice. TSPO is a well validated biomarker of neuroinflammation, and it is upregulated in glial cells in response to brain insults and neurodegeneration (35-38, 77). We measured increased TSPO levels in the CB of male and female Slc39a14-KO mice relative to WT. Both male and female KO mice exhibited an increase in TSPO levels across the CB cortex and CN. Although an increase in TSPO levels is indicative of ongoing neuroinflammation and neurodegeneration, it does not reveal cytoarchitectural changes in the affected brain regions. Therefore, to further delineate the neuropathological changes in the CB of Slc39a14-KO mice we performed immunohistochemistry using the microglia marker Iba-1 (39).

Microglia are the resident macrophages of the brain. An increase in microglial number and/or a shift towards phagocytic morphology are indicative of a neuroinflammatory response to neurodegeneration (39, 54). We discovered a significant increase in microglia number in deep cerebellar regions (DCR) of several CB lobules, indicative of ongoing neurodegeneration. These neuropathological changes have never been reported in the context of elevated Mn concentrations in the CB, or in the SLC39A14 mutation carriers. However, the observed and quantified increase in microglia in the CB of Slc39a14-KO mice does not specify the neuronal populations undergoing neurodegeneration. Furthermore, while most of the DCR across both sexes exhibit markedly increase microglial clustering and area coverage, there appear to be sex-specific regional differences in microglial activation, especially in the CN (Figure 5A-D). These changes may be due to sex-specific differences in the levels of metals accumulation or increased susceptibility to Mn neurotoxicity in the males. Quantification of microglial activation using the machine-learning imaging application IMARIS demonstrated a marked shift in resting microglia to a phagocytic activated phenotype in the CB of Slc39a14-KO mice, expressed as a decrease in microglial perimeter (Figure 5E-H).

To delineate susceptible neuronal populations in the DCR of the CB, we performed cCASP3 immunohistochemistry and discovered a significant increase in the number of cCASP3 positive PCs in male and female PN60 Slc39a14-KO mice. The findings from cCASP3 staining and quantification indicates that in conjunction with the RNAseq data, microglial activation and elevated TSPO levels, PCs in the CB of Slc39a14-KO mice appear to be undergoing cell death via apoptosis. Furthermore, some of the cCASP3 can be found within the BG. BG have been demonstrated to express cCASP3 which is non-lethal (54). Although the same group that discovered the non-lethal expression of cCASP3 in BG postulates that in an in-vitro model cCASP3 expression is used for cellular turnover, the underlying reason for in-vivo remains to be elucidated. Potentially some of the cCASP3 expression in the BG can be explained by BG turnover as has been noted in the developing brain (55, 56). Furthermore, BG have been shown to be responsible for Ca²⁺ and K⁺ homeostasis in the PCL (57). BG guide the radial migration of GCs and layering of GCs within the GCL (58). While the layering of GCs is not necessarily strict in nature, GC comprise a large portion of excitatory input into the PCs and are the most numerous neuronal subtypes in the brain (58). Thus, dysfunction in the BG may result in altered or delayed migration of differentiated GCs, producing altered signaling to the PCs later in life (59). Additionally, H&E staining of formalin fixed, and paraffin embedded (FFPE) CB sections revealed that PCs of Slc39a14-KO mice have an eosinophilic and pyknotic appearance, indicative of ongoing neurodegeneration (Figure 6E-F; Supplementary Figure 5).

The defects in physiological tremor, lack of coordination and gait, and dystonic phenotype observed in the Slc39a14-KO mice implicates compromised PCs output and altered baseline CN firing properties (24, 43). Here, we demonstrate population-wide reduction in CN firing rate and increased variability within-population of PCs simple spike and CN firing properties in the CB of Slc39a14-KO mice (Figures 7 and 1). PCs generate two types of action potentials: simple spikes and complex spikes. The generation of each spike type is influenced by a combination of intrinsic and extrinsic factors (60-64). As we have found alterations only in simple spikes, it is possible that mechanisms and anatomy that produce complex spikes are unaffected or normalized in young adult Slc39a14-KO mice while the mechanisms of simple spike production may have varying degrees of dysregulation within the population of PCs in the CB of Slc39a14-KO mice. It is intriguing that the only population-wide change in activity was found in CN neurons. Abnormalities in the firing of CN cells are sufficient to generate robust motor phenotypes and correcting CN firing can improve motor deficits (24, 43, 65). Importantly, the Slc39a14-KO mice have a dystonic phenotype that appears during development. Another mouse model with severe dystonia and developmental onset similarly demonstrated healthy PC simple spike activity and abnormal nuclei neuron activity in adulthood. However, these mice displayed both PC and nuclei neuron firing abnormalities during development (43). It is therefore possible that the insults in Slc39a14-KO mice which cause dystonia during development may similarly result in altered PC activity that is later corrected (or compensated for) and additionally cause insurmountable circuit alterations in the CN and potentially elsewhere in the motor circuit leading to a lifetime of dystonia.

One such extracerebellar area of interest is the striatum. Several publications including those from our lab, demonstrate that elevated Mn concentrations produce inhibition of DA release in the striatum (2, 13, 78-79). Whether this effect is unique to DA and to the striatum alone, remains to be answered. However, inhibition of striatal DA release may be a downstream effect of CB dysfunction, either due to ongoing abnormal CN neuron signaling (Figure 1) or alternations to the motor circuit established during development (67). Disynaptic anatomical (68) and rapid electrophysiological (69) connections have been established between the CN and the striatum. This is further supported by a recent publication by Washburn and colleagues (20) demonstrating that striatal DA release and activity of SNpc dopaminergic neurons is directly modulated by glutamatergic innervation from the CN. This is a critical finding as it bridges the gap between the marked inhibition of striatal DA release and the CB pathology observed in the Slc39a14-KO mice.

While we have demonstrated alterations in the principal cells of the CB, PCs and nuclei neurons, we have not explored potential other changes in the CB microcircuitry. The CB cortex consists of three cellular layers: molecular, PC, and granular layers. These layers contain the PCs and seven distinct types of interneurons including basket cells, Lugaro cells, Golgi cells, stellate cells, granule cells, candelabrum cells and unipolar brush cells (27, 58, 70-71). Along with climbing fiber inputs from the inferior olivary nucleus and mossy fibers from the pontine nucleus, these interneurons collectively modulate PC firing and, consequently, CN firing. Within PCs, nuclei neurons, and various interneuron types there are known subpopulations that may confer varying susceptibility to Mn toxicity or KO effects (subpopulations within the CB reviewed in 72).

In summary, CB neuropathology has received little to no attention in the field of Mn neurotoxicity. The current work aims to expand the scope of our studies to the CB to include the analysis of neuronal and glial cell types in the context of neuropathological changes induced by Mn hyperaccumulation in this animal model of the human disease. Finally, a recent publication examining the effects of chronic Mn exposure in welders which are occupationally exposed to elevated levels of Mn in the welding fumes (72) indicates that the CB is a target for Mn neurotoxicity in the human brain and support our current findings in this animal model of Mn-induced neurotoxicity in SLC39A14 mutation carriers.

Materials and Methods:

Animal husbandry and genotyping:

Animal studies were reviewed and approved by the Florida International University Animal Care and Use Committee, comply with the ARRIVE guidelines, and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Slc39a14-KO founder mice were generously provided by Dr. Robert J. Cousins from the University of Florida, where the colony was originally described. Briefly and as described in the original publication, Zip14-/+ and Zip14+/+ founder mice were obtained through a contract with the Mutant Mouse Regional Resource Center at the University of California, Davis. A targeted mutation in Zip14 gene (exons 3–5) was generated in strain 129/SvEvBrd derived embryonic stem cells. The chimeric mice were bred to C57BL/6J albino mice to generate Zip14+/− mice. Zip14−/− mice were obtained through further breeding of founder Zip14+/− mice at the University of Florida. Animals were housed on a 12:12 light:dark cycle with ad-libitum food and water access. Animals were bred using 1:1 male:female heterozygous mice with litters being weaned at post-natal day (PN) 21. A small tail clip was obtained during animal weaning at post-natal day 21 using aseptic conditions. The tissue was homogenized and treated with DNA extract solution, followed by PCR master-mix and primers. A more detailed description of the protocol can be found here (2).

Physiological tremor recordings:

Tremor recordings were performed using San Diego Instruments Tremor Monitoring System. All tremor recordings were performed after 7pm, during the dark cycle. Animals were placed into the recording chambers for habituation for 15 minutes, followed by a 30-minute recording session.

ICP-MS Metals Analysis in Cerebellum:

Tissue was harvested and immediately frozen on dry ice, with subsequent storage at −80°C until further analysis. Briefly, the CB was suspended in 1 mL of concentrated nitric acid and incubated inside a heat block at 95°C for 2 hours. Internal standard mixture was added, and total volume was made up to 10ml, using 3% nitric acid. Germanium was used as the internal standard. The resulting solution was injected directly into the ICP-MS (2).

Immunohistochemistry:

Animals were perfused according to our standardized protocol, previously published here (2). The tissue was sectioned in the sagittal plane and, using pertinent brain atlases, every 3^rd section was pulled for immunohistochemical assessment. Briefly, the tissue was rinsed in pH7.4, room temperature PBS, exposed to peroxidase blocking and antigen-retrieval solutions, followed by incubation in the blocking solution and in the primary anti-Iba-1 antibody (GeneTex; GTX635400; 1:1000) solution for 24 hours at room temperature. Next, the tissue was exposed to biotinylated secondary antibodies and avidin-biotin-peroxidase complex. A solution containing 3,3’-diaminobenzidine tetrachloride was used as the chromogen, with exposure of 3 min at RT. Omission of primary antibody was used as the negative control. All steps were performed on a shaker. Images were captured using the Keyence BZ-X800 system for the Iba-1 and cCASP3 quantification.

For immunofluorescence the protocol was identical to that of the DAB protocol, with the omission of the peroxidase blocking and antigen retrieval. After blocking the tissue was incubated in the primary antibody solution containing cCASP3 antibody (GeneTex; GTX86952; 1:100) for 48hrs at 4C. Following the primary antibody incubation, the tissue was exposed to secondary Donkey anti-Rabbit Alexa 488 solution (1:200) for an hour at RT. Representative images used for cCASP3 were captured using Olympus Fluoview FV10i confocal system. No primary control was used to set maximum laser intensity settings. Images were captured as a 20-layer thick z-stacks and are shown as projections of the entire stack.

FFPE tissue processing was done as follows: animals underwent standardized transcardial perfusion as described above, however, the perfusion was done using only PBS. The brains were harvested and transferred into ice-cold 4% PFA solution, followed by paraffin embedding cassettes and then processed using the “Routine Surgical” protocol in the Epredia Revos tissue processor. For the H&E staining the tissue was embedded into paraffin and sectioned at 5μm. Vector Laboratories H&E kit (H-3502) was utilized for the staining following the manufacturer’s guidelines.

RNA isolation and analysis:

Briefly, the freshly dissected CB were flash frozen on dry ice until ready for RNA isolation. To isolate the RNA, we homogenized the weighed samples in 1ml of TRIZOL per 100mg of tissue, using a powered tissue homogenizer. Following the homogenization step, 0.2ml chloroform was added per 1mL of TRIZOL. The chloroform/TRIZOL solution was vortexed for 15s and incubated at RT for 3min. The samples were then centrifuged at 12,000g for 15min at 4C. Following the separation, the top layer containing the aqueous phase was transferred into a clean tube, 0.5ml of isopropyl alcohol per 1mL of TRIZOL used in the initial step was added to induce RNA precipitation. Afterwards the samples were kept at RT for 10min and then centrifuged at 12,000g for 10min at 4C. Following the second centrifugation, the supernatant was removed, leaving the RNA pellet. The pellet was washed with 75% ethanol, using 1:1 ratio of 75% ethanol to TRIZOL volume. The RNA pellet was then gently resuspended in 75% ethanol, and centrifuged at 7,500g for 5min at 4C. The ethanol washing step was then repeated, the final ethanol wash was removed, and the pellet was dried for 7min and then redissolved in DEPC-treated water. The isolated and purified RNA was checked for concentration and purity and shipped to Novogene Co., Ltd. for sequencing and analysis. GO analysis data were plotted utilizing the FDR corrected p-values obtained from Novogene analysis. Sequencing was carried out using the Illumina 6000 platform. Once sequenced, the data was transformed to raw reads, utilizing CASAVA base calling. After controlling for sequencing errors and GC content distribution, the data were filtered to remove adapter contamination, uncertain and low-quality nucleotides. Reads were aligned using the HISAT2 algorithm. Data were processed using the DESeq2 software. Negative binomial distribution model was used to calculate the p-values. FDR was calculated using Benjamini-Hochberg method. Volcano plot data were graphed for both, corrected and uncorrected p-values. Upregulated DEGs are color-coded as red, while downregulated DEGs are blue; significantly DEGs after FDR correction are color coded in their respective color scheme and outlined in black. Log2 fold change was set at 1; p-values were converted into −log10 values with 1.3 line on the y-axis representing the uncorrected p-value of 0.05.

TSPO Quantitative Autoradiography:

Brains from naïve animals were extracted, immediately flash frozen on dry ice, and stored at −80°C until further assessment. The tissue was cryosectioned in coronal plane at 20μm and sequentially mounted onto poly-l-lysine coated slides. Once mounted, the slides were dried at 37°C for 30 minutes and stored at −80°C. [³H]-DPA-713 radioligand was utilized for unbiased quantitative autoradiography of TSPO. Briefly, matching tissue slides were selected for specific and non-specific solution incubations, containing 1nM [³H]-DPA-713 or 1nM [³H]-DPA-713 and 10μM PK11195, respectively. Once exposed to the washing buffers, tissue slides were incubated in ligand-containing buffers, washed, allowed to dry overnight and exposed onto Kodak MR film for 4-6 weeks. Image capturing and analysis were performed using MCID Core 7.1.

Quantification of Iba-1 percent area coverage and cCASP3 cell numbers:

Once coverslipped, the slides were imaged on the Keyence BZ-X800 microscope. For quantification purposes Iba-1 images were captured using the 10x objective in best focus mode and then stitched together to obtain a high-resolution image. For cCASP3 the images were captured as 20-layer thick z-stacks using the 20x objective and were subsequently stitched together. Iba-1 areas of interest were identified using the Allen brain atlas and approximate tracings of activated regions in the DCR of the Slc39a14-KO mice were identified in the matched sex and age WT controls. The image threshold was obtained through ImageJ by identifying the smallest distinguishable microglia fragment in the B&W mode. Following the threshold application, the analysis function was used to obtain signal per ROI. Data represent a combination of %-area covered, as analyzed across 6 sections starting from 0.42mm laterally in 1:3 series. For cCASP3 total length of each CB lobule was obtained. The cCASP3 expression was quantified within the DCR, in the matching regions of Iba-1 immunoreactivity. Thus, the length of the CB lobules was measured to 50% from the DCR. The obtained distance was recorded in mm and the number of cCASP3 cells in the PCL was counted along each side of every lobule to match the designated DCR border.

Quantification of microglial morphology using IMARIS:

IMARIS-based perimeter quantification was carried out in DAB immunostained sections used for %-area coverage analysis. Images were captured as z-stacks of 18-21 images and stitched into MIP projections, converted into IMARIS file format for downstream analysis. For segmentation and machine learning a selection of cells was picked to train the algorithm. Cell selection ranged from cells occupying centers of microglial clusters, three neuronal layers within the CB cortex and white matter tracts to account for variable levels of background staining, segmentation within the clusters and identification of thin microglial projections. ML algorithms were trained on each animal individually. For quantification, 10 cells were selected per CB lobule and circularity and area values were collected for each individual cell. Since circularity is a function of area and perimeter, perimeter calculations were carried out to represent microglial projection complexity; that is – the more ramified the microglia, the larger the perimeter values and the more phagocytic, the smaller. Values were plotted and analyzed for each lobule independently. For perimeter analysis no multiple comparison’s corrections were carried out.

Synchrotron X-ray fluorescence microscopy (XFM) and imaging

Cryo-sectioning:

Three Slc39a14-KO male mice and three age-matched control mice brains were used for this experiment. Brains were sectioned using a Shandon cryotome, whose chamber was maintained at a temperature of −16°C. Frozen brains were allowed to rest in the cryochamber until thermal equilibrium was attained. Following this, the tissue was embedded on cryocassette using a generous amount of Optimal Cutting Temperature (OCT) compound, mounted onto the cryostage, cut at thickness of 10 μm sagittal using a blade. Sections of interest (containing CB) were identified by comparing cresyl-violet stained sections to the Allen brain atlas. Samples for XFM were mounted on ultralene film glued to kodak frames, stored at a temperature of −80° C until used as samples for XFM imaging.

Technique:

Synchrotron X-ray fluorescence imaging data were collected at two different beamlines. Samples were allowed to thaw at room temperature for 30 minutes before exposing to X-rays. Regions of interest from samples (CB), identified using visible light optical microscope, were transferred on 5x5mm Silicon Nitrile window frames. Optical microscopy images were collected to measure the coordinates of PCL within the CB for navigation during XFM.

9-ID Bionanoprobe, Advanced Photon Source, Argonne National Laboratory 46:

Samples, set at an angle of 15° to the incident beam of X-ray, were exposed to an X-ray beam of 10.5 keV at room temperature. Samples were raster scanned in fly-scan mode, and x-ray fluorescence spectra were recorded using a silicon drift detector Vortex ME-7, 7-element detector, mounted perpendicularly to the incident beam. The elemental densities were quantified using standard RF8-200-S2453 by AXO. The MAPS program was used to analyze and fit the collected data on a per-pixel basis. The MAPS program and ImageJ were used to visualize the fitted data (74).

3-ID, Hard X-ray Nanoprobe, National Synchrotron Light source -II, Brookhaven National Laboratory:

The samples were measured in the He environment at room temperature at 250 mmHg pressure. An X-ray beam of 10.5 keV was focused by a Fresnel zone plate with outer diameter of 30 nm. The specimen was placed at 15° relative to the incident beam, facing a 3-element silicon drift detector (Vortex ME3) which was mounted perpendicular to the incident X-ray beam. The specimen was raster scanned and collected fluorescence spectrum was processed using the PyXRF program on per-pixel basis (75). The XFM maps were quantified using the normalized fluorescence spectrum obtained from a multi-element calibration standard (Micromatter, 41149), measured under the same conditions as the sample.

X-Ray Fluorescence Data Analysis:

X-ray fluorescence spectra recorded by the detectors were normalized to the respective intensities of the incident X-ray beam. The spectra were fitted, thereby obtaining integrated normalized fluorescence intensities (counts/s), which in turn were converted into the units of μg/cm² using fitted fluorescence spectra of the standards with known concentration. The standards used at Argonne National Lab are selected in a way to ensure two or more elements from the sample are present in the standards (Ca, Fe, and Cu). The fitted fluorescence spectrum of the standard generates a calibration curve providing a calibration ratio for each element to convert the values in counts/s to μg/cm². Although the quantification method in APS-MAPS and PyXRF differed slightly, both adhere to the theory of quantitative fluorescence analysis. To quantify Mn, Fe, Cu and Zn, elements with the closest atomic numbers Mn, Ga, Cu and Co were used, thereby ensuring calibration ratio of S, and matching the respective elements from the standards. To report the results in the units to μg of the elements per gram of wet tissue, the value of density of water was used to obtain a scaling factor of 0.001. To report the results in μM concentration, a factor of 18.2 was used. Concentration of all elements of interest within discovered Mn accumulations is obtained by selecting the pixels of interest using ImageJ. (See Supplementary Tables 1, 4 and 5).

Statistical Analysis:

Statistical analysis was performed using Prism 10.2.2. The α level was set as 0.05. Normality was assessed using the Shapiro-Wilk test. Tremors data were analyzed using mixed effects model with FDR adjustment, corrections across multiple comparisons were performed using the two-stage step-up method of Benjamini, Krieger and Yekutieli. ICP-MS, quantitative TSPO autoradiography and microglial area coverage data were analyzed using Welch’s t-test. Microglial perimeter and cCASP3 data were analyzed using one-way ANOVA with LSD or Bonferroni correction for multiple comparisons, respectively.

Surgery for in-vivo awake electrophysiology:

Adult mice aged two to five months of both sexes were prepared for awake electrophysiology recordings via the surgical implantation of a headplate and the opening of a ~2 mm diameter craniotomy. These surgical techniques have been described in detail previously (37). In short, mice were provided with preemptive analgesics including slow-release buprenorphine (1 mg/kg) and meloxicam (2 mg/kg) subcutaneously. Mice were anesthetized with isoflurane, the surgical site was cleaned of fur and sterilized, and an incision was made over the skull extending anterior to Bregma and posterior to the occipital plate. The skull was cleaned of fascia and the craniotomy site was placed 6.4 mm posterior and 1.3-1.5 mm lateral from Bregma. After performing the craniotomy, the exposed brain was immediately covered in antibiotic ointment. A custom-made metal headplate was affixed anterior to the craniotomy site and a custom-made plastic chamber was affixed surrounding the craniotomy site using Metabond (Parkell; C&B Metabond Quick Adhesive Cement System) followed by dental cement (A-M Systems; dental cement powder #525000 and solvent #526000). The chamber was sealed with Kwik-Sil silicone (WPI; Kwik-Sil). Mice were allowed to recover for three to five days before recordings were made during which they were provided with meloxicam (2 mg/kg) as postoperative analgesia for at least the first three days.

In-vivo awake electrophysiology recordings:

Mice were acclimated to the head fixation apparatus before recordings, which were performed while the mice were awake and restfully standing head-fixed atop a wheel. The implanted headplate was screwed into a metal frame to minimize head movements. Then the silicone cap and antibiotic ointment were removed from the recording chamber. The chamber was then filled with sterile saline (0.9% sodium chloride) for the duration of the recording. Recordings were made of both PCs and CB interposed nucleus cells. PCs were identified by their anatomical depth and the presence of both simple and complex spikes. CN cells were identified by their anatomical location and lack of complex spikes. Recordings were performed using tungsten electrodes (Thomas Recording; ~8 MΩ) that were positioned using a micromanipulator (Sutter Instrument Co.; MP-225). Signals were amplified and bandpass filtered between 0.3–13 kHz (NPI Electronic Instruments; ELC-03XS). Signals were then digitized (CED; Power 1401) before being recorded with Spike2 software (CED). Voltage signals were also passed through an audio monitor (Grass Technologies; AM10) to allow for auditory spike detection and localization by the experimenter during recordings.

Analysis of in-vivo awake electrophysiology:

Spikes were sorted from raw voltage traces using Spike2 software (CED) and spike times were imported into MATLAB (MathWorks) and Excel (Microsoft) for further analysis using custom scripts. Analyzed recordings were ~60 s duration (61) and only stable and well-isolated cells were considered for analysis. Spike times were used to determine firing rate, coefficient of variance (CV), and CV2 (41). Firing rate was defined as the number of spikes that occurred over the sampled duration (spikes/second). CV was calculated by dividing the standard deviation of the interspike intervals (ISIs) by the average ISI. CV2 was calculated using the following equation: $\frac{2\mid IS{I}_n-IS{I}_{n-1}\mid }{IS{I}_n-IS{I}_{n-1}}$. Prism software (Graphpad) was used for identification of outliers and performing statistics. Data from males and females were visualized and then pooled for further analysis due to consistent effect trends for each sex per genotype. Outliers were identified and removed using the ROUT method (Q = 0.1%). Differences between means were tested for significance using Welch’s t-tests followed by family-wise Holm-Šídák adjustments of p values. Differences between variances were tested for significance using F-tests followed by family-wise Holm-Šídák adjustments of p values. Adjusted p values less than 0.05 were considered significant.

Visualization of in-vivo awake electrophysiology:

Graphs of electrophysiology data were generated using Prism (Graphpad) software and Adobe Illustrator software. Raw voltage traces were generated in Spike2 software and exported to Adobe Illustrator for visualization. Schematics were generated using Adobe Illustrator software. The number of animals included in analyses is represented by “N” and the number of cells included in analyses is represented by “n”. P values ≤0.05 are indicated with an asterisk.

Figure 8. Electrophysiological analysis of cerebellar nuclei cell activity in awake adult Slc39a14-KO mice.

A) Schematic of extracellular recordings of cerebellar nuclei cells in adult mice. B-C) Representative 1-second-long raw nuclei cell spike traces from WT (B) and KO (C) mice. Scale = 100 ms. T-test and F-test p values are listed above each comparison. Asterisk indicates adjusted p value ≤ 0.05. D-I) Analysis of nuclei cell spike activity from adult PN64-161 mice. WT N = 10 and n = 27 (female N = 7 and n = 19), KO N = 9 and n = 25 (female N = 7 and n = 18). D-F) Cerebellar nuclei cell firing rate (D), CV (E), and CV2 (F). WT mean ± SEM is 78.37 ± 4.094 (D), 0.4011 ± 0.02044 (E), and 0.3835 ± 0.01588 (F), respectively. KO mean ± SEM is 62.01 ± 3.492 (D), 0.4885 ± 0.03573 (E), and 0.4332 ± 0.02464 (F), respectively.

Highlights:

- Slc39a14-knockout mice, a preclinical model of childhood-onset manganese-induced dystonia-parkinsonism, express high levels of manganese in the cerebellum.

- This murine model exhibits deficits in physiological tremor consistent with cerebellar dysfunction.

- RNA sequencing of the cerebellum revealed upregulation in genes of immune response and cell death.

- Neuroinflammation and neurodegeneration markers confirmed the RNA sequencing results and revealed Purkinje cell degeneration.

- Electrophysiological studies of cerebellum neurocircuitry discovered altered firing patterns.

Data availability statement

All of the raw RNA sequencing has been uploaded and is publicly available from the Gene Expression Omnibus. The GSE number is 303733.