Age-dependent loss of primary cilia in cholinergic interneurons in caudate putamen (CPu) of G2019S-LRRK2-KI mice.

(A) Confocal images of cholinergic interneurons from aged wildtype (wt) and G2019S-LRRK2-KI (G2019S) mice stained for neuronal ciliary marker adenylate cyclase 3 (AC3), choline acetyltransferase (ChaT) and DAPI. Scale bar, 10 μm. (B) Quantitation of ciliation in ChaT+ and ChaT-neurons in CPu from young (4-5 months) wt and G2019S mice. Ciliation in ChaT+ neurons was also quantified separately from dorsomedial (DM) and ventrolateral (VL) CPu. (C) Ciliation in ChaT+ and ChaT-neurons in middle-aged (13-14 months) wt and G2019S mice. (D) Ciliation in aged (18-24 months) mice. Each datapoint is from an individual mouse, analyzing 2 sections per animal and 100-200 ChaT+ and 400-800 ChaT-cells. Bars represent mean ± s.e.m (n=4, 2 males and 2 females per age group and genotype). Significance was determined by unpaired two-tailed student’s T-test; **p<0.01; ***p<0.001; ****p<0.0001. (E) Quantitation of ciliary length in ChaT+ neurons from aged (18-24 months old) wt and G2019S mice. Bars represent mean ± s.e.m (n=3); *p<0.05.

Age-dependent loss of primary cilia in cholinergic neurons in globus pallidus (GP) of G2019S-LRRK2-KI mice.

(A) Confocal images of cholinergic neurons in GP from aged wildtype (wt) and G2019S-LRRK2-KI (G2019S) mice stained for neuronal ciliary marker adenylate cyclase 3 (AC3), choline acetyltransferase (ChaT) and DAPI. Scale bar, 10 μm. (B) Quantitation of ciliation in ChaT+ and ChaT-neurons in CPu from young (4-5 months), middle-aged (13-14 months) and aged (18-24 months) wt and G2019S mice. Each datapoint is from an individual mouse, analyzing 2 sections per animal and 100-200 ChaT+ and 400-800 ChaT-cells. Bars represent mean ± s.e.m (n=4, 2 males and 2 females per age group and genotype). Significance was determined by unpaired two-tailed student’s T-test; **p<0.01.

Ciliary deficits in forebrain cholinergic neurons already in young G2019S-LRRK2-KI mice.

(A) Confocal images of cholinergic neurons in nucleus basalis of Meynert (NBM) from aged wt and G2019S mice stained for neuronal ciliary marker adenylate cyclase 3 (AC3), choline acetyltransferase (ChaT) and DAPI. Scale bar, 10 μm. (B) Quantitation of ciliation in ChaT+ and ChaT-neurons in NBM from young (4-5 months), middle-aged (13-14 months) and aged (18-24 months) wt and G2019S mice. (C) Confocal images of cholinergic neurons in medial septum (MS) from aged wt and G2019S mice stained for neuronal ciliary marker adenylate cyclase 3 (AC3), choline acetyltransferase (ChaT) and DAPI. Scale bar, 10 μm. (D) Quantitation of ciliation in ChaT+ and ChaT-neurons in MS/diagonal band (DB) from young (4-5 months), middle-aged (13-14 months) and aged (18-24 months) wt and G2019S mice. Each datapoint is from an individual mouse, analyzing 2 sections per animal and 100-200 ChaT+ and 400-800 ChaT-cells. Bars represent mean ± s.e.m (n=4, 2 males and 2 females per age group and genotype). Significance was determined by unpaired two-tailed student’s T-test; ***p<0.001; ****p<0.0001.

Age-dependent loss of primary cilia in brainstem cholinergic neurons in G2019S-LRRK2-KI mice.

(A) Confocal images of cholinergic neurons in pedunculopontine nucleus (PPN) from aged wt and G2019S mice stained for neuronal ciliary marker adenylate cyclase 3 (AC3), choline acetyltransferase (ChaT) and DAPI. Scale bar, 10 μm. (B) Quantitation of ciliation in ChaT+ neurons in PPN from young (4-5 months), middle-aged (13-14 months) and aged (18-24 months) wt and G2019S mice. (C) Confocal images of cholinergic neurons in laterodorsal tegmental nucleus (LDTgN) from aged wt and G2019S mice stained for neuronal ciliary marker adenylate cyclase 3 (AC3), choline acetyltransferase (ChaT) and DAPI. Scale bar, 10 μm. (D) Quantitation of ciliation in ChaT+ neurons in LDTgN from young (4-5 months), middle-aged (13-14 months) and aged (18-24 months) wt and G2019S mice. Each datapoint is from an individual mouse, analyzing 2 sections per animal and 100-200 ChaT+ cells. Bars represent mean ± s.e.m (n=4, 2 males and 2 females per age group and genotype). Significance was determined by unpaired two-tailed student’s T-test; *p<0.05; **p<0.01; ***p<0.001.

Basal forebrain cholinergic neurons from G2019S-LRRK2-KI mice show unique pRab12 staining.

(A) Confocal images of cholinergic neurons in horizontal diagonal band (HDB) from young, middle-aged and aged wt and G2019S mice stained with antibodies against phospho-Rab12 (pRab12), choline acetyltransferase (ChaT) and DAPI. Scale bar, 10 μm. (B) Quantification of pRab12-positive structures in ChaT+ neurons in HDB, MS and NBM from young adult and 13+ months old wt and G2019S mice. Data are from young adult (n=2) and 13+ months old wt (n=4) and G2019S (n=5) mice, with 50-100 ChaT+ neurons from 2 independent sections quantified per animal. (C) Quantification of pRab12 fluorescence intensity in ChaT+ neurons in HDB, MS and NBM from young adult and 13+ months old wt and G2019S mice. Integrated density from young adult (n=2) amd 13+ months old wt (n=4) and G2019S (n=5) mice, with 20-45 ChaT+ neurons from 2 independent sections quantified per animal. Significance was determined by unpaired two-tailed student’s T-test; ***p<0.001; ****p<0.0001.

Early dystrophic changes in cholinergic axons derived from basal forebrain cholinergic neurons in G2019S-LRRK2-KI mice.

(A) Confocal images of dorsal hippocampal CA1/2 regions from young and aged wt and G2019S mice stained for vesicular acetylcholine transporter (VAChT) and DAPI. Scale bar, 100 μm. (B) Manual quantitation of dystrophic axonal clusters in hippocampal CA1/CA2 regions from young (4-5 months) and 13+ months old wt and G2019S mice. (C) Quantitation of dystrophic axons > 3 μm2 in hippocampal CA1/CA2 regions from young (4-5 months) and 13+ months old wt and G2019S mice. Box and whisker plots (5-95 percentile) are from 5 animals (male and female) each per age and genotype, with three sections per animal and three images per section quantified. Significance was determined by unpaired two-tailed student’s T-test; *p<0.05; **p<0.01; ****p<0.0001.

Age-dependent loss of cholinergic innervation derived from brainstem cholinergic neurons in G2019S-LRRK2-KI mice.

(A) Confocal images of thalamic dorsal lateral geniculate nucleus (dLGN) from aged wt and G2019S mice stained for vesicular acetylcholine transporter (VAChT) and DAPI. Scale bar, 30 μm. (B) Quantitation of innervation density in dLGN from young (4-5 months) and 13+ months old wt and G2019S mice. Box and whisker plots (5-95 percentile) are from 5 animals (male and female) each per age and genotype, with three sections per animal and five images per section quantified. Significance was determined by unpaired two-tailed student’s T-test; **p<0.01; ***p<0.001.

Age-dependent loss of ChaT+ cell counts in nucleus basalis of Meynert in G2019S-LRRK2-KI mice.

(A) ChaT+ cell counts in CPu from aged wt and G2019S mice. Bars represent mean ± s.e.m (n=4, 2 males and 2 females per genotype). (B) ChaT+ cell counts in GP from aged wt and G2019S mice. (C) ChaT+ cell counts in brainstem LDTgN from aged wt and G2019S mice. (D) Confocal images of NBM from aged wt and G2019S mice stained for ChaT and DAPI, with Bregma coordinates indicated to the left. Scale bar, 100 μm. (E) Quantitation of ChaT+ cell counts in NBM from young (4-5 months) and 13+ months old wt and G2019S mice. ChaT+ cells were counted from seven serial 30 μm coronal sections across the NBM. Bars represent mean ± s.e.m (n=4 for young adult, n=8 for 13+ months). Significance was determined by unpaired two-tailed student’s T-test; ***p<0.001.

Age-dependent cholinergic cell loss in diagonal band in G2019S-LRRK2-KI mice.

(A) Confocal images of horizontal diagonal band (HDB) from aged wt and G2019S mice stained for ChaT, p75 and DAPI, with Bregma coordinates indicated to the left. Scale bar, 100 μm. (B) Quantitation of ChaT+ cell counts in medial septum (MS), vertical diagonal band (VDB) and horizontal diagonal band (HDB) from young adult (4-5 months) and 13+ months old wt and G2019S mice. ChaT+ cells were counted from fifteen alternate serial 30 μm coronal sections across the MS, VDB and HDB. Bars represent mean ± s.e.m (n=4 for young adult, n=8 for 13+ months). Significance was determined by unpaired two-tailed student’s T-test; ****p<0.0001.

Age-dependent cholinergic cell loss in pedunculopontine nucleus in G2019S-LRRK2-KI mice.

(A) Confocal images of pedunculopontine nucleus (PPN) from aged wt and G2019S mice stained for ChaT, nNOS and DAPI, with Bregma coordinates indicated to the left. Scale bar, 100 μm. (B) Quantitation of ChaT+ cell counts in rostral PPN, caudal PPN and total PPN from young adult (4-5 months) and 13+ months old wt and G2019S mice. ChaT+ cells were counted from fifteen alternate serial 30 μm coronal sections across the PPN (total PPN), as well as from the first six alternate serial rostral sections or the last six alternate serial or caudal sections, respectively. Bars represent mean ± s.e.m (n=4 for young adult, n=8 for 13+ months). Significance was determined by unpaired two-tailed student’s T-test; ***p<0.001; ****p<0.0001.

Regional sampling parameters

Normal ciliation in calretinin-positive and parvalbumin-positive interneurons in CPu of aged G2019S-LRRK2-KI mice.

(A) Confocal images of calretinin-positive interneurons in CPu from aged wt and G2019S mice stained for AC3, calretinin and DAPI. Scale bar, 10 μm. (B) Quantitation of ciliation in calretinin-positive (calret+) interneurons in CPu from aged (18-24 months) wt and G2019S mice. (C) Confocal images of parvalbumin-positive interneurons in CPu from aged wt and G2019S mice stained for AC3, parvalbumin and DAPI. Scale bar, 10 μm. (D) Quantitation of ciliation in parvalbumin-positive (parv+) interneurons in CPu from aged (18-24 months) wt and G2019S mice. Each datapoint is from an individual mouse, analyzing 2 sections per animal and 100-120 calret+ or parv+ cells, respectively. Bars represent mean ± s.e.m (n=3).

Cholinergic neurons in brainstem and basal ganglia from G2019S-LRRK2-KI mice do not show staining with pRab12 antibody.

(A) Confocal images of cholinergic neurons in pedunculopontine nucleus (PPN) from young, middle-aged and aged wt and G2019S mice stained with antibodies against phospho-Rab12 (pRab12), choline acetyltransferase (ChaT) and DAPI. (B) Cholinergic neurons in LDTgN from aged wt and G2019S mice stained as above. (C) Cholinergic neurons in GP from aged wt and G2019S mice stained as above. (D) Cholinergic neurons in CPu from aged wt and G2019S mice stained as above. All scale bars are 10 μm.

pRab12 staining is phospho-state-specific and vesicular in nature.

(A) Confocal images of cholinergic neurons in VDB from middle-aged G2019S mice either with or without pretreatment with phosphatase (PPase) before staining with antibodies against phospho-Rab12 (pRab12), choline acetyltransferase (ChaT) and DAPI. Scale bar, 10 μm. (B) Confocal image of cholinergic neuron in HDB from middle-aged G2019S mouse stained with pRab12 antibody and DAPI after an antigen retrieval protocol. Arrow points to pRab12-positive vesicular structures. Scale bar, 10 μm.

Dystrophic axons derived from basal forebrain cholinergic neurons in G2019S-LRRK2-KI mice stain positive for both VAChT and ChaT.

Confocal images of hippocampal CA1/2 regions from young wt and G2019S mice stained for VAChT, ChaT and DAPI. Insert shows colocalization of both cholinergic markers. Scale bar, 100 μm.

Cholinergic innervation in dLGN from G2019S-LRRK2-KI mice as assessed by both VAChT and ChaT staining.

Confocal images of dLGN from aged wt and G2019S mice stained for VAChT, ChaT and DAPI. Scale bar, 30 μm.

Cholinergic neurons in medial septum and diagonal band are positive for p75.

(A) Confocal images of different brain areas from 13-months old wt mice stained for ChaT, p75 and DAPI, with Bregma coordinates indicated to the left. Scale bar, 100 μm. (B) Quantification of ChaT+ neurons co-stained with p75 from different brain aras as indicated in middle-aged wt and G2019S mice. Colocalization was scored from 50-100 cholinergic neurons per brain area and genotype.

Brainstem cholinergic neurons in PPN and LDTgN are positive for nNOS.

(A) Confocal images of different brain areas from 13-months old wt mice stained for ChaT, nNOS and DAPI, with Bregma coordinates indicated to the left. Scale bar, 100 μm. (B) Quantification of ChaT+ neurons co-stained with nNOS from different brain aras as indicated in middle-aged wt and G2019S mice. Colocalization was scored from 50-100 cholinergic neurons per brain area and genotype.

Cholinergic cell counts in HDB.

ChaT+ and p75+ cell counts in alternate sections through the horizontal diagonal band from young adult (4-5 months), middle-aged (13-14 months) and aged (18-24 months) male and female mice as indicated.

Cholinergic cell counts in VDB.

ChaT+ and p75+ cell counts in alternate sections through the vertical diagonal band from young adult (4-5 months), middle-aged (13-14 months) and aged (18-24 months) male and female mice as indicated.

Cholinergic cell counts in MS.

ChaT+ and p75+ cell counts in alternate sections through the medial septum from young adult (4-5 months), middle-aged (13-14 months) and aged (18-24 months) male and female mice as indicated.

Cholinergic cell counts in PPN.

ChaT+ and nNOS+ cell counts in alternate sections through the PPN from young adult (4-5 months), middle-aged (13-14 months) and aged (18-24 months) male and female mice as indicated.