Early lysosome defects precede neurodegeneration with amyloid-β and tau aggregation in NHE6-null rat brain

Abstract

Loss-of-function mutations in the X-linked endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome in males. Christianson syndrome involves endosome dysfunction leading to early cerebellar degeneration, as well as later-onset cortical and subcortical neurodegeneration, potentially including tau deposition as reported in post-mortem studies. In addition, there is reported evidence of modulation of amyloid-β levels in experimental models wherein NHE6 expression was targeted. We have recently shown that loss of NHE6 causes defects in endosome maturation and trafficking underlying lysosome deficiency in primary mouse neurons in vitro. For in vivo studies, rat models may have an advantage over mouse models for the study of neurodegeneration, as rat brain can demonstrate robust deposition of endogenously-expressed amyloid-β and tau in certain pathological states. Mouse models generally do not show the accumulation of insoluble, endogenously-expressed (non-transgenic) tau or amyloid-β. Therefore, to study neurodegeneration in Christianson syndrome and the possibility of amyloid-β and tau pathology, we generated an NHE6-null rat model of Christianson syndrome using CRISPR-Cas9 genome-editing. Here, we present the sequence of pathogenic events in neurodegenerating NHE6-null male rat brains across the lifespan. NHE6-null rats demonstrated an early and rapid loss of Purkinje cells in the cerebellum, as well as a more protracted neurodegenerative course in the cerebrum. In both the cerebellum and cerebrum, lysosome deficiency is an early pathogenic event, preceding autophagic dysfunction. Microglial and astrocyte activation also occur early. In the hippocampus and cortex, lysosome defects precede loss of pyramidal cells. Importantly, we subsequently observed biochemical and in situ evidence of both amyloid-β and tau aggregation in the aged NHE6-null hippocampus and cortex (but not in the cerebellum). Tau deposition is widely distributed, including cortical and subcortical distributions. Interestingly, we observed tau deposition in both neurons and glia, as has been reported in Christianson syndrome post-mortem studies previously. In summary, this experimental model is among very few examples of a genetically modified animal that exhibits neurodegeneration with deposition of endogenously-expressed amyloid-β and tau. This NHE6-null rat will serve as a new robust model for Christianson syndrome. Furthermore, these studies provide evidence for linkages between endolysosome dysfunction and neurodegeneration involving protein aggregations, including amyloid-β and tau. Therefore these studies may provide insight into mechanisms of more common neurodegenerative disorders, including Alzheimer's disease and related dementias.

Keywords: amyloid beta; lysosomes; neurodegeneration; rat model; tau.

Figure 1

Generation and validation of NHE6-null rats. (A) Two single guide RNAs (sgRNA) were inserted into exon 7 of the endogenous Slc9a6 gene. (B) Schematic representation of the targeted Slc9a6 locus harbouring the 2-bp (TT) insertion. The insertion generated a premature stop codon. (C) Sequences of wild-type (WT) and NHE6-null rats. Genomic DNA sequence, isolated from rat tail biopsy, shows the sequence in the wild-type rat and the 2-bp insertion causing a premature stop codon in NHE6. (D) The mRNA level of NHE6 in wild-type and NHE6-null rat brain was measured by quantitative real-time PCR. Two different sets of primers were used to validate the mRNA level. The mRNA level was normalized against the reference gene. Two-tailed unpaired t-test with Welch’s correction was used (P < 0.0001 for both primer sets, n = 3 for each genotype). (E) Absence of NHE6 protein in NHE6-null rat brain. Immunoprecipitation (IP) of NHE6 in the whole brain lysates from wild-type and NHE6-null rats validates the absence of NHE6 protein. Actin was used as a loading control. IgG Heavy chain was detected at ∼50 kDa after IP with NHE6 antibody. (F) Validation of NHE6 protein absence by immunofluorescence. Brain sections from wild-type and NHE6-null rats at 2 months were stained with NHE6 antibody. Scale bar = 500 µm.

Figure 2

Brain length and size analysis of NHE6-null rats across the lifespan. (A) Representative images of wild-type (WT) and NHE6-null male rats at 3, 6, 12 months (m). Scale bar = 500 µm. The length of the anterior-posterior (A-P) axis was measured at the interhemisphere divided from the tip of cerebrum to the end of cerebellum (CB) as indicated in the panel. The dotted lines indicate the measured area of the CTX (yellow) and cerebellum (magenta). (B) The A-P length of NHE6 was shorter than wild-type at 3 months (P < 0.0001). The length of wild-type and NHE6-null rat brain continued to increase until 9 months (P < 0.0001). At 12 months, the A-P length of NHE6-null rat brain was significantly decreased compared to that of 12-month-old wild-type (P = 0.0006; two-tailed unpaired t-test for each time point). (C) The CTX area of NHE6-null decreased at 12 months compared to that of wild-type (two-tailed unpaired t-test with Welch’s correction, P = 0.0009). (D) The cerebellum area of NHE6-null was smaller than that of wild-type across the lifespan (P = 0.0044 for 3 months, P = 0.0002 for 9 months, P < 0.0001 for 12 months, two-tailed unpaired t-test with Welch’s correction). The number of animals was for B–D: 3-month-old wild-type  = 8, 3-month-old Null = 7, 9-month-old wild-type  = 6, 9-month-old Null = 6, 12-month-old wild-type  = 7, 12-month-old Null = 8. Data are presented as mean ± SEM. Asterisks represent P-values as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001.

Figure 3

Early cerebellar degeneration. (A) Sagittal cerebellar sections and quantification from 2-month-old wild-type (WT) and NHE6-null rats were stained with a Purkinje cell (PC) marker, calbindin along with DAPI. The staining of calbindin reduced in NHE6-null rats. Scale bar = 500 µm. The number of Purkinje cell cells was manually counted and divided by area (µm²). Two-tailed unpaired t-test with Welch’s correction was performed (P < 0.0001). (B) Cerebellar sections and its quantification from wild-type and NHE6-null rats at 2 months were stained with calbindin and GM2. GM2/calbindin-positive cells were detected only in NHE6-null rats. Scale bar = 10 µm. The quantification shows the per cent of area covered by GM2 (P < 0.0001). (C) Cerebellar sections were stained with p62 and calbindin. NHE6-null rats at 2 months showed increases in p62 staining (P = 0.02). (D) Cerebellar sections were stained with ubiquitin (Ub) and calbindin. The staining of Ub was increased in NHE6-null rats at 2 months (P = 0.0113). Two-tailed unpaired t-test with Welch’s correction was performed. Values from each brain section (three sections/each animal) are clustered in different colour codes according to each animal and plotted as a small dot. Means from each biological replicate are overlaid on the top of the full dataset as a bigger dot. P-value and standard error of the mean (SEM) were calculated using values from all sections from all animals (wild-type = 3, Null = 3) used in biological replicates. All data are presented as mean ± SEM. (E) Bielschowsky’s silver staining of cerebellum at 12 months. The staining in the paramedian lobule of cerebellum from NHE6-null rats prominently decreased compared to wild-type. Scale bar = 100 µm. (F) Nissl staining of paramedian lobule of cerebellum at 12 months. Arrows indicate Purkinje cell cells. The loss of Purkinje cell cells was observed in NHE6-null rats. Insets showed diffusing Nissl staining of Purkinje cell cells in NHE6-null rats. Scale bar = 100 µm. (G) Haematoxylin and eosin staining of wild-type and NHE6-null rats at 12 months. Multifocal vacuolization (red arrow) was observed in the paramedian lobule of cerebellum along with gliosis (black arrow) in NHE6-null rats. Scale bar = 100 µm.

Figure 4

Lysosomal defects in NHE6-null rats. (A) GM2 ganglioside staining in the CA1 and CA3 of wild-type (WT) and NHE6-null rats at 3 months. Coronal brain sections were stained with GM2 (magenta) along with NeuN (green). In the CA1 and CA3 region, GM2 prominently accumulates in neurons of NHE6-null rats compared to wild-type. White boxes in the merged images indicate the location of magnified GM2 images. Scale bar = 500 µm; scale bar for magnified images = 50 µm. (B) GM2 staining in basolateral amygdala (BLA) region of wild-type and NHE6-null rats at 3 months. Brain sections were stained with GM2 (magenta) and NeuN (green). GM2 was detected in neurons in NHE6-null rats. The GM2 staining was prominently detected in neurons in NHE6-null rats. Arrows indicates GM2-accumulating neurons. Insets show GM2-positive neuron cells. Scale bar = 500 µm; scale bar for insets = 50 µm. (C) Quantification of GM2 covered area (%) in CA1 (P < 0.0001), CA3 (P < 0.0001) and BLA (P = 0.0006) regions of wild-type and NHE6-null rats from 3 and 18 months. Two-way ANOVA was performed followed by Tukey’s honestly significant difference (HSD) (wild-type  = 3, Null = 3 for each time point). For BLA, unpaired t-test with Welch’s correction was conducted. (D) Lamp1 (lysosomal marker) staining with NeuN in wild-type and NHE6-null rats at 3 months and 18 months. The Lamp1 staining in the CA1 region and other regions increases in neurons of NHE6-null rats compared to wild-type. Scale bar= 50 µm. (E) Quantification of Lamp1 covered area (%) at 3 and 18 months (P < 0.0001). Two-way ANOVA was conducted followed by Tukey’s HSD (wild-type = 3, Null = 3 for each time point). Values from each brain section (three sections/each animal) are clustered in different colour codes according to each animal and plotted as a small dot. Means from each biological replicate are overlaid on the top of the full dataset as a bigger dot. P-value and SEM were calculated using values from all sections from all animals used in biological replicates. All data are presented as mean ± SEM.

Figure 5

Autophagy dysfunction occurs after lysosome defects in NHE6-null rats. (A) p62 staining in wild-type (WT) and NHE6-null rats at 18 months. The p62 staining was detected in the hippocampus (HP), CTX and corpus callosum (CC) region of NHE6-null rats. Scale bar = 500 µm. (B) Magnified images of p62 staining in the retrosplenial (RSP) CTX and CC in wild-type and NHE6-null rats at 18 months. Scale bar = 20 µm. (C) Ub staining in wild-type and NHE6-null rats at 18 months. Neuronal Ub inclusion bodies were detected in the RSP CTX and CC of NHE6-null rats. The Ub staining was observed in the CC region. Scale bar = 20 µm. (D) LC3 staining in wild-type and NHE6-null rats at 18 months. Numbers of LC3 puncta were increased in the RSP CTX (P = 0.0011) and CC (P = 0.0055) of NHE6-null rats. (E) Quantification of p62 and Ub covered area (%) in the CC and CTX regions at 3 and 18 months (P < 0.0001). Two-way ANOVA was conducted followed by Tukey’s HSD (wild-type  = 3, Null = 3 for each time point). The number of LC3 puncta was counted at 18 months (P = 0.0011 for CTX, P = 0.0055 for CC). Two-tailed unpaired t-test with Welch’s correction was performed (wild-type = 3, Null = 3). All data are presented as mean ± SEM. Asterisks represent P-values as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001.

Figure 6

Neuronal loss and axonal pathology in NHE6-null rats. (A) Representative images of 12-month-old wild-type (WT) and NHE6-null rat brain sections stained with Nissl. The overall brain size of NHE6-null rat decreased. Enlarged ventricles and loss of piriform/entorhinal CTX were observed. Boxes indicate the location of the magnified hippocampal images. In the CA1 region, the Nissl staining decreased in NHE6-null rats. Scale bar for the whole brain image = 500 µm; magnified image = 200 µm. Quantification for volumes of the hippocampus (HP), the piriform/entorhinal CTX, and the lateral ventricle, cortical layer thickness and dentate gyrus thickness is presented in Supplementary Fig. 3. (B) Haematoxylin and eosin staining in the CA3 region of HP from 12-month-old wild-type and NHE6-null rats. Multifocal vacuolization was observed in NHE6-null rats. Scale bar = 20 µm. Arrows indicate vacuoles. (C) Representative images of NeuN staining. Brain sections from wild-type and NHE6-null rats at 3 and 12 months were stained with a neuronal marker, NeuN. White boxes indicate the location of magnified images. Scale bar for the whole hippocampal image = 200 µm; magnified images = 50 µm. (D) Quantification of number of NeuN-positive cells in the CA1 region of wild-type and NHE6-null rats at 3 and 12 months. At 12 months, the number of NeuN-positive cells decreased in NHE6-null rats compared to 12-month-old wild-type and 3-month-old NHE6-null rats. Two-way ANOVA was performed followed by Tukey’s HSD (wild-type  = 5, Null = 5 for each time point). Tukey’s adjusted P = 0.0062 (Null at 3 months versus Null at 14 months), P = 0.0028 (wild-type at 12 months versus Null at 12 months). Values from each brain section (four sections/each animal) were clustered in different colour codes according to each animal and plotted as a small dot. Means from each biological replicate are overlaid on the top of the full dataset as a bigger dot. P-value and SEM were calculated using values from all sections from all animals used in biological replicates. All data are presented as mean ± SEM. Asterisks represent P-values as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001. (E) Bielschowsky’s silver staining of 12-month-old wild-type and NHE6-null rats. The staining of cortical region and major axonal tracks including corpus callosum (CC) are reduced in NHE6-null rats. Boxes indicate the location of the magnified cortical images. In CTX, the staining was barely detected in NHE6-null rats. Thinning of CC was observed in NHE6-null rats. Scale bar for the whole brain image = 500 µm; magnified image= 200 µm. (F) Haematoxylin and eosin staining of 12-month-old wild-type and NHE6-null rats. Multifocal vacuolization of the CC and adjacent grey matter was observed in NHE6-null rats. Insets represent the magnified image of vacuolization. Scale bar = 200 µm.

Figure 7

Tau-associated pathologies in the aged brains of NHE6-null rats. (A) Brain tissue from NHE6 wild-type (WT) and null rats at 3 months and 18 months were sequentially extracted in TBS, sarkosyl-containing buffer and urea-containing buffer. The whole lanes of AT8 and TAU5 were used for quantification. Representative western blot images of tau fractionation are shown. For quantification of tau fractionation, densitometry of AT8 and TAU5 was measured, using the signal present in the entire lane. Data are expressed as AT8 signal relative to TAU5 signal. The insoluble AT8 fraction increased in NHE6-null rats compared to wild-type at 18 months (P = 0.0027 for wild-type versus Null at 18 months, P = 0.00034 for Null at 3 versus 18 months). Two-way ANOVA with Tukey’s HSD was performed (wild-type = 5, Null = 5 for each time point). (B) AT8 immunostaining in the HP and CTX region at 18 months. The overall increase in AT8 staining was exhibited in NHE6-null rats. Scale bar = 500 µm. (C) PHF1 and NeuN staining in the SN region of wild-type and NHE6-null rats at 18 months. PHF1-positive NeuN staining was observed in NHE6-null rats. Scale bar = 20 µm. (D) AT8 and GFAP staining in the CC region of wild-type and NHE6-null rats at 18 months. AT8 and GFAP-positive staining was profoundly detected in NHE6-null rats. Arrow indicates AT8/GFAP-positive stained cells. Scale bar = 20 µm. (E) ThioflavinS (ThioS) staining from wild-type and NHE6-null rats at 18 months. Prominent ThioflavinS staining was detected in the CA1, CC and SN regions of NHE6-null rats. (F) Quantification of AT8 covered area (%) in the CC (P = 0.0009), SN (P = 0.0008) and CA1 (P < 0.0001) regions of wild-type and NHE6-null rats at 3 and 18 months was calculated. Two-way ANOVA followed by Tukey’s HSD was conducted. Also, ThioflavinS covered area (%) n the CC (P = 0.0007), SN (P = 0.0011) and CA1 (P = 0.0072) at 18 months was quantified. Two-tailed unpaired t-test with Welch’s correction was performed. Values from each brain section (three sections/each animal) are clustered in different colour codes according to each animal and plotted as a small dot. Means from each biological replicate are overlaid on the top of the full dataset as a bigger dot. P-value and SEM were calculated using values from all sections from all animals (wild-type  = 3, Null = 3) used in biological replicates. All data are presented as mean ± SEM. Data are presented as mean ± SEM. Asterisks represent P-values as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 8

Aggregation of amyloid-β in NHE6-null rats without an increase in overall amyloid-β level. (A–C) The level of amyloid-βs including amyloid-β₃₈, amyloid-β₄₀ and amyloid-β₄₂ did not change from wild-type (WT) and NHE6-null rats at 18 months. (D–F) GuHCl-soluble fractions in all amyloid-β, amyloid-β₄₀ and amyloid-β₄₂ from NHE6-null rats increased while TBS-soluble fractions decreased. (G–I) A proportion of GuHCl-soluble fraction increases while TBS-soluble fraction decreases in all amyloid-β, amyloid-β₄₀ and amyloid-β₄₂. (J) Ratio of amyloid-β_42/40 increase in GuHCl-soluble fraction (wild-type = 5, Null = 5). All statistical analysis details described in detail in the main text and Supplementary Table 3. (K and L) APP/amyloid-β staining in the CC and CTX region of wild-type and NHE6-null rats at 18 months. Increase in 6 ×10¹⁰ and fibrillar amyloid-β oligomer (clone: OC) was detected in NHE6-null rats. Two-tailed unpaired t-test with Welch’s correction was performed (wild-type = 3, Null = 3). Values from each brain section (three sections/each animal) are clustered in different colour codes according to each animal and plotted as a small dot. Means from each biological replicate are overlaid on the top of the full dataset as a bigger dot. P-value and SEM were calculated using values from all sections from all animals used in biological replicates. All data are presented as mean ± SEM. Data are presented as mean ± SEM. Asterisks represent P-values as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.