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A cell autonomous torsinA requirement for cholinergic neuron survival and motor control

  1. Samuel S Pappas
  2. Jay Li
  3. Tessa M LeWitt
  4. Jeong-Ki Kim
  5. Umrao R Monani
  6. William T Dauer  Is a corresponding author
  1. University of Michigan, United States
  2. Columbia University Medical Center, United States
Research Advance
Cite this article as: eLife 2018;7:e36691 doi: 10.7554/eLife.36691
3 figures, 6 tables and 1 additional file


Figure 1 with 5 supplements
Conditional cholinergic neuron deletion of torsinA causes cell autonomous loss of striatal cholinergic neurons.

(A) Unilateral stereological quantification of the number of ChAT-positive neurons in the striatum of ChAT-CKO and control mice (One-way ANOVA F(3,28) = 3.589, p=0.02, Dunnett’s multiple comparisons test: adjusted p value = 0.049; ‘WT’=Tor1aFlx/+; ‘Cre Control’=ChAT-Cre+, Tor1a Flx/+; ‘Het Control’=Tor1 aFlx/-; ‘ChAT-CKO’=ChAT-Cre+, Tor1aFlx/-). (B) ChAT immunohistochemistry of coronal sections containing dorsal striatum from WT and ChAT-CKO mice (cc = corpus callosum). (C) Percent reduction in cell density by striatal quadrant (DL = dorsolateral; DM = dorsomedial, VL = ventrolateral, VM = ventromedial). (D) Significant ChI loss is selective for dorsal striatal quadrants. Cell density quantification in control and ChAT-CKO striatal quadrants (Two-way ANOVA main effect of genotype F(3,112) = 24.02, p<0.0001; main effect of quadrant F(3,112)=8.398, p<0.0001; interaction F(9,112)=8.11, p<0.0001. Post-hoc Tukey’s multiple comparisons test). (E) Basal forebrain neurons are spared in ChAT-CKO mice. Stereological quantification of P75-immunoreactive basal forebrain cholinergic neurons in the nucleus basalis of meynert (NBM), medial septum/nucleus of the vertical limb of the diagonal band (MS/VDB), and globus pallidus (GP). No differences in the number of cholinergic neurons was observed (NBM, t(13)=1.684, p=0.11; MS/VDB, t(13)=1.537, p=0.148; GP, t(13)=0.5, p=0.625). (F) P75 immunohistochemistry of sagittal sections containing basal forebrain cholinergic neuron populations. i.c. = internal capsule, ST = striatum.

Figure 1—figure supplement 1
ChAT-Cre is expressed prenatally.

(Upper panels) ChAT-Cre mice were crossed with Ai14 Cre reporter mice. Offspring were collected immediately after birth, brain sections were generated and observed under epifluorescence microscopy. (Lower panels) adjacent sections costained for torsinA. Scale bar = 50 μm.

Figure 1—figure supplement 2
Independent cohort confirmation of selective striatal cholinergic neuron loss in ChAT-CKO mice.

(A) Bilateral unbiased stereology of ChAT-immunoreactive neurons in the dorsal striatum (t(12)=4.42, p=0.0008). (B) Unbiased stereology of parvalbumin (PV) immunoreactive neurons in the dorsal striatum (t9 = 0.699, p=0.50). (C) Unbiased stereology of Nissl positive cells in the dorsal striatum (Welch’s t-test; t7.803=0.655, p=0.53).

Figure 1—figure supplement 3
ChAT-positive neurons are reduced in a topographic pattern throughout the rostrocaudal extent of the dorsal striatum.

Significant decreases in ChAT-positive cells were observed in the dorsolateral and dorsomedial segments of the dorsal striatum (dorsolateral striatum, two-way ANOVA main effect of genotype F(3,156)=74.77, p<0.0001, main effect of rostrocaudal section, F(5,156)=10.07, p<0.0001, no interaction F(15,156)=1.204, p=0.273; Dorsomedial striatum main effect of genotype F(3,156)=50.01, p<0.0001, main effect of rostrocaudal section, F(5,156)=41.81, p<0.0001, no interaction F(15,156)=1.646, p=0.067. Post-hoc Tukey’s test was performed for all significant main effects. * represents significant difference between ChAT-CKO mice and all control groups). No significant reductions were observed in the ventral segments of the dorsal striatum (ventrolateral; genotype F(3,156)=2.84, p=0.039 [post-hoc Tukey’s test adjusted p=0.129 or higher for all comparisons], rostrocaudal section F(5,156)=0.479, p=0.79, interaction F(15,156)=0.249, p=0.99. ventromedial; genotype F(3,156)= 2.706, p=0.047 [post-hoc Tukey’s test adjusted p=0.107 or higher for all comparisons], rostrocaudal section F(5,156)=46.28 p<0.0001, interaction F(15,156)=0.672, p=0.80).

Figure 1—figure supplement 4
Time course of torsinA protein loss in dorsal and ventral striatum.

(A,B) TorsinA and ChAT staining in dorsal and ventral striatum brain sections from P0 ChAT-CKO and control mice. (C) TorsinA mean fluorescence intensity analysis in dorsal or ventral striatal ChI (Two-way ANOVA main effect of genotype F1,195= 85.67, p<0.0001; Region, n.s., F1,195=3.301, p=0.07; interaction F1,195=21.34, p<0.0001. Sidak’s multiple comparisons test, Dorsal striatum control vs ChAT-CKO, p=0.003, Ventral striatum control vs ChAT-CKO, p<0.0001, ChAT-CKO dorsal striatum vs ventral striatum, p=0.0002; Control dorsal striatum vs ventral striatum, n.s., p=0.199). (D,E) Frequency histograms of torsinA mean fluorescence intensity in dorsal (n = 57 control, n = 54 ChAT-CKO neurons) and ventral striatal ChI (n = 52 control, n = 36 ChAT-CKO neurons). Scale bar = 10 µm.

Figure 1—figure supplement 5
Time course of torsinA protein loss in basal forebrain.

(A) TorsinA and ChAT staining in basal forebrain brain sections from P0 ChAT-CKO and control mice. (B) TorsinA mean fluorescence intensity analysis of control and ChAT-CKO (Welch’s t-test t138.2=17.35, p<0.0001) and frequency histograms of torsinA mean fluorescence intensity in basal forebrain cholinergic neurons ChI (n = 91 control, n = 79 ChAT-CKO neurons). Scale bar = 10 µm.

Figure 2 with 1 supplement
ChAT-CKO mice have significantly fewer brainstem and spinal cord cholinergic neurons.

(A,B) Stereological quantification of ChAT-positive or NeuN-positive neurons in the pedunculopontine nucleus (PPN) of control and ChAT-CKO mice (ChAT; t(14)=4.531, p=0.0005. NeuN; t(14)=0.095, p=0.92). (C,D) Stereological quantification of ChAT-positive or NeuN-positive neurons in the laterdorsal tegmental nucleus (LDT) of control and ChAT-CKO mice (ChAT; t(14)=3.571, p=0.003. NeuN; t(14)=1.934, p=0.073). (E,F) Quantification of the number of ChAT-positive neurons in the cervical spinal cord of control and ChAT-CKO mice (t(6)=3.654, p=0.0107). Scale bars = 100 μm.

Figure 2—figure supplement 1
Absence of gliosis in the brainstem of ChAT-CKO mice.

Immunohistochemistry of GFAP (glial fibrillary acidic protein; specific to astrocytes) and Iba-1 (ionized calcium-binding adapter molecule 1; specific to microglia) in sagittal sections of control and ChAT-CKO hindbrain demonstrate normal distribution of glia.

Figure 3 with 4 supplements
Motor behavior is severely disrupted in ChAT-CKO mice.

(A) Representative image of a control and ChAT-CKO mouse demonstrates severe kyphosis and unkempt coat. (B) ChAT-CKO mice exhibit significantly increased kyphotic curvature during locomotion (Mann-Whitney U = 35, p<0.0001). (C) ChAT-CKO mice exhibit a significantly reduced latency to fall during forelimb suspension (Mann-Whitney U = 71.5, p<0.0001). (D, E) ChAT-CKO mice are hypoactive in the open field (horizontal movement, t(23)=2.345, p=0.028; vertical rears, welch-corrected t(15.1) = 2.345, p=0.033). (F) Performance on the accelerated rotarod does not significantly differ from controls (two-way repeated measures ANOVA, genotype, F(1,43)=0.75, p=0.389; trial, F(9,387)=55.63, p<0.0001; interaction, F(9,387)=1.194, p=0.297). (G - I) ChAT-CKO mouse gait is abnormal during locomotion (paw angle, two-way ANOVA main effect of genotype, F(1,56)=30.54, p<0.0001, main effect of limb F(3,56)=51.02, p<0.0001, interaction F(3,56)=13.51, p<0.0001, post-hoc Sidak’s multiple comparisons test. Stance width, t(14)=3.329, p=0.005. Stride length, two-way ANOVA genotype F(1,28)=3.164, p=0.086, limb F(1,28)=0.02, p=0.887, interaction F(1,28)=0.0001, p=0.989).

Figure 3—figure supplement 1
Representative examples of control and ChAT-CKO spinal cords demonstrate significant kyphotic curvature.
Figure 3—figure supplement 2
ChAT-CKO mice are significantly hypoactive.

(Upper panel) Open field analysis of horizontal movements (two-way repeated measures ANOVA main effect of genotype F(1,23)=5.498, p=0.02, time F(11,253)=9.222, p<0.0001, interaction F(11,253)=0.899, p=0.541, post-hoc Sidak’s multiple comparisons test). (Lower panel) Open field analysis of vertical rearing movements (two-way repeated measures ANOVA main effect of genotype F(1,23)=4.413, p=0.046, time F(11,253)=2.452, p=0.0063, interaction F(11,253)=0.987, p=0.458).

Figure 3—video 1
Representative video demonstrating tremulousness, kyphosis, and hyperactivity in ChAT-CKO mice, as compared to controls.
Figure 3—video 2
ChAT-CKO exhibit twisting and tremulousness, but not limb clasping during tail suspension.

Representative video demonstrating tail suspension test in control and ChAT-CKO mice.



Table 1
Behavioral properties of Dlx-CKO and ChAT-CKO mice.
Motor functionDlx-CKOChAT-CKO
Pappas et al., 2015 eLife 4:e08352present manuscript
Tail suspensionTrunk twistingTrunk twisting
Forelimb clasping-
Hindlimb clasping-
Open fieldHyperactivityHypoactivity
Response to handlingExaggeratedReduced
Weakness, latency to fallGrid hang reductionWire hang reduction
GaitNormal by eyeAbnormal by eye
Slightly reduced stance widthIncreased stance width
-Increased paw angle
Overt postural abnormalities-Severe kyphosis
Tremulous movement-Severe
Labored breathing-Severe
Table 2
Vulnerability of cholinergic populations.

(*)=Unconfirmed by independent marker.

Cre expressionCell death vulnerability
Cholinergic populationDlx-CreChAT-CreDlx-CreChAT-Cre
Dorsolateral striatum
(including dorsal caudate
Dorsomedial striatum
(including ventral caudate
Nucleus accumbensConfirmedConfirmed--
Basal forebrainConfirmedConfirmedSparedSpared
Cholinergic BrainstemAbsentConfirmedn/aSevere (*)
Primary Motor NeuronsAbsentConfirmedn/aModerate
Table 3
Properties of cholinergic neuronal populations.

‘Nucleus Basalis Complex’=Nucleus Basalis of Meynert, Horizontal limb of the diagonal band of Broca, Ventral Pallidum, Magnocellular Preoptic Area, Substantia Inominata, Nucleus of the Ansa Lenticularis. ‘Septa”l = Medial Septum, Vertical Limb of the Diagonal Band of Broca. ‘Cholinergic Brainstem’=Pedunculopontine Nucleus, Laterodorsal Tegmental Nucleus (Pappas et al., 2015; Mena-Segovia and Bolam, 2017; Gonzales and Smith, 2015; Manns et al., 2000; Unal et al., 2012; Petzold et al., 2015; Kanning et al., 2010; Kreitzer, 2009; Zaborszky et al., 2012; Garcia-Rill, 1991; Semba et al., 1988; Semba and Fibiger, 1992; Phelps et al., 1990a; Phelps et al., 1988; Phelps et al., 1990b; Phelps et al., 1989; Aroca and Puelles, 2005; Schambra et al., 1989).

Cholinergic populationNeuronal classFiring propertiesEfferent projectionsAfferent inputsBirth date/final mitosisEmbryonic originChAT expression
Dorsolateral striatum (including dorsal caudate putamen)Interneurontonically active, 2–10 Hz baseline firing rateLocal - striatal spiny projection neurons and fast spiking interneuronsThalamus, sensorimotor cortex, striatal spiny projection neurons, striatal interneuronsE12-E15MGE~E16
Dorsomedial striatum (including ventral caudate putamen)Interneurontonically active, 2–10 Hz baseline firing rateLocal - striatal spiny projection neurons and fast spiking interneuronsThalamus, association cortices, striatal spiny projection neurons, striatal interneuronsE12-E15MGE~E16
Nucleus accumbensInterneurontonically active, 0.6–12 Hz baseline firing rateLocal - striatal spiny projection neurons and fast spiking interneuronsThalamus, frontal cortex, striatal spiny projection neurons, striatal interneuronsE12-E15MGE~E16
Basal forebrainProjection neuronTonic/burst, subtype dependentCortex (Nucleus Basalis Complex), Hippocampus (Septal)Medulla, locus ceruleus, substantia nigra, ventral tegmental area, hypothalamic nuclei, nucleus accumbens, amygdala, local intrinsic GABAergic and glutamatergic collateralsE11-E15POA/MGE~E15-16
Cholinergic BrainstemProjection neuronepisodicMidbrain, superior colliculus, thalamus, globus pallidus, hypothalamus, septum, striatum, cortexBrainstem reticular formation, midbrain central gray, lateral hypothalamus-zona incerta, cortex, amygdala, basal forebrain, basal ganglia output nuclei, brainstem and spinal cord sensory nucleiE12-E13Ventral rhombomere 1 (r1)
Primary Motor NeuronsProjection neuronsubtype dependentMuscleMotor Cortex, local spinal cord interneurons and sensory neuronsE11-E12Ventral spinal cord progenitor domainsE13
Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
(Mus musculus)
Tor1aNANCBI Gene: 30931;
Encodes TorsinA
Strain, strain
background(M. musculus)
ChAT-CreJackson LaboratoriesStock ID 006410Chattm2(cre)Lowl; (Chat-IRES-Cre)
Strain, strain
background(M. musculus)
Tor1aFlx/FlxJackson LaboratoriesStock ID 025832Tor1atm3.1Wtd
Strain, strain
background(M. musculus)
Tor1a-/-Jackson LaboratoriesStock ID 006251Tor1atm1Wtd
Millipore AB144PRRID: AB_20797511:100
AntibodyP75 Neurotrophin
Santa Cruz sc6188RRID: AB_22672541:100
AntibodyNeuNCell Signaling #12943RRID: AB_26303951:500
AntibodyGFAPCell Signaling #3670PRRID: AB_5610491:1000
AntibodyIba-1Wako 019–19741RRID: AB_8395041:500
AntibodyTorsinAAbcam ab34540RRID: AB_22407921:100
RRID: AB_1625421:800
RRID: AB_25357921:800
RRID: AB_1625431:800
RRID: AB_25358531:800
RRID: AB_23403961:800
Commercial assay
or kit
Vector LaboratoriesPk-6100Vectastain elite
ABC kit
Table 4
Antibodies used for immunohistochemistry.
PrimaryCholine AcetyltransferaseGoat-1:100Millipore AB144P
PrimaryP75 Neurotrophin ReceptorGoat-1:100Santa Cruz sc6188
PrimaryNeuNRabbit-1:500Cell Signaling #12943
PrimaryGFAPMouse-1:1000Cell Signaling #3670P
PrimaryIba-1Rabbit-1:500Wako 019–19741
PrimaryTorsinARabbit-1:100Abcam ab34540
Secondaryanti-mouseDonkeyAlexafluor-6471:800ThermoFisher A-31571
Secondaryanti-rabbitDonkeyAlexafluor-4881:800ThermoFisher A-21206
Secondaryanti-rabbitDonkeyAlexafluor-5551:800ThermoFisher A-31572
Secondaryanti-goatDonkeyAlexafluor-5551:800ThermoFisher A-21432
Secondaryanti-goatDonkeybiotin1:800Jackson Immunoresearch 705-065-003
Table 5
Stereology parameters.
RegionMarkerCounting frame (μm)Grid size (μm)Guard zone (μm)Dissector (μm)Section cut thickness (μm)
StriatumChAT100 × 100250 × 25011040
NBMP7590 × 90200 × 20053050
MS/VDBP7590 × 90200 × 20053050
GPP75100 × 100140 × 14053050
PPN and LDTChAT75 × 75150 × 15053050
PPN and LDTNeuN40 × 40250 × 25053050

Data availability

All data generated during this study are included in the manuscript and supporting files

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