Fig 2: Distinct lysosomal phenotypes among different types of neurons in the cortex (CTX) in young Tmem106b-/- mice. a, b Immunostaining of Calbindin and Cath D in brain sections from 5-month-old WT and Tmem106b-/- mice, and images were captured from the frontal cortex. The intensity of Cath D in Calbindin-positive neurons was quantified in b. n = 3. **, p < 0.01, unpaired t-test. Scale bar = 10 µm. c, d Immunostaining of PVALB and Cathepsin D (Cath D) in brain sections from 5-month-old WT and Tmem106b-/- mice. Representative images from the frontal cortex are shown. The intensity of Cath D in PVALB-positive interneurons was quantified in d. n = 3. **, p < 0.01, unpaired t-test. Scale bar = 10 µm. e, f Immunostaining of CUX1, Cath D, and NeuN in brain sections from 5-month-old WT and Tmem106b-/- mice. Representative images from the frontal cortex are shown. The intensity of Cath D in CUX1-positive excitatory neuron was quantified in f. n = 3. ****, p < 0.0001, unpaired t-test. Scale bar = 10 µm

Fig 3: Degeneration of Purkinje cells in aged Tmem106b-/- mice. a, b Cerebellar sections from 16-month-old WT and Tmem106b-/- mice were stained with anti-calbindin antibody and Hoechst. Purkinje cell numbers in the anterior lobe (AL) and other regions (Others) were quantified in b. Scale bar = 100 µm. n = 3–4, ***, p < 0.001, unpaired t-test. c, d Cerebellar sections from 16-month-old WT and Tmem106b-/- mice were immuno-stained with antibodies of calbindin and parvalbumin (PVALB). Zoom-in images show PVALB-positive interneurons in the MCL adjacent to the region with Purkinje cell loss. The number of PVALB-positive interneurons in the molecular layer of the anterior lobe (AL) was quantified in d. Scale bar = 100 µm. n = 3, ***, p < 0.001, unpaired t-test. e, f Western blot analysis of the protein level of Calbindin, NeuN, and TMEM106B in 16-month-old WT and Tmem106b-/- cerebellar lysates. GAPDH was used as an internal control. Relative protein levels were quantified in f. n = 5, ***, p < 0.001, unpaired t-test

Fig 4: Distinct lysosomal phenotypes among different types of neurons in the cerebellum of young Tmem106b-/- mice. a, b Immunostaining of PVALB and Cathepsin D (Cath D) in cerebellar sections from 2-month-old WT and Tmem106b-/- mice. Representative images from the molecular cell layer are shown. The intensity of Cath D in PVALB-positive interneurons was quantified in b. n = 3. ****, p < 0.0001, unpaired t-test. Scale bar = 10 µm. c, d Immunostaining of Cathepsin D (Cath D) and Hoechst in cerebellar sections from 2-month-old WT and Tmem106b-/- mice, and images were captured from granule cell layer. The intensity of Cath D in the granule cell layer was quantified in d. n = 3. **, p < 0.001, unpaired t-test. Scale bar = 10 µm. e, f Immunostaining of Cathepsin D (Cath D) and MAP2 in cerebellar sections from 2-month-old WT and Tmem106b-/- mice. Representative images from the DCN region are shown. The intensity of Cath D in MAP2-positive DCN neurons was quantified in f. Zoom-in image shows the enlarged lysosomes in 2-month-old Tmem106b-/- mice compared with WT mice. n = 3. ****, p < 0.0001, unpaired t-test. Scale bar = 10 µm