1. Neuroscience

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
https://doi.org/10.7554/eLife.36691
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Cite this article
  1. Samuel S Pappas
  2. Jay Li
  3. Tessa M LeWitt
  4. Jeong-Ki Kim
  5. Umrao R Monani
  6. William T Dauer
(2018)
A cell autonomous torsinA requirement for cholinergic neuron survival and motor control
eLife 7:e36691.
https://doi.org/10.7554/eLife.36691
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3 figures, 6 tables and 1 additional file

Figures

Figure 1 with 5 supplements
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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 …

https://doi.org/10.7554/eLife.36691.002
Figure 1—figure supplement 1
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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. …

https://doi.org/10.7554/eLife.36691.003
Figure 1—figure supplement 2
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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 …

https://doi.org/10.7554/eLife.36691.004
Figure 1—figure supplement 3
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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)=7…

https://doi.org/10.7554/eLife.36691.005
Figure 1—figure supplement 4
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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 …

https://doi.org/10.7554/eLife.36691.006
Figure 1—figure supplement 5
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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=17.3…

https://doi.org/10.7554/eLife.36691.007
Figure 2 with 1 supplement
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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, …

https://doi.org/10.7554/eLife.36691.008
Figure 2—figure supplement 1
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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 …

https://doi.org/10.7554/eLife.36691.009
Figure 3 with 4 supplements
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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 …

https://doi.org/10.7554/eLife.36691.010
Figure 3—figure supplement 1
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Representative examples of control and ChAT-CKO spinal cords demonstrate significant kyphotic curvature.
https://doi.org/10.7554/eLife.36691.011
Figure 3—figure supplement 2
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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, …

https://doi.org/10.7554/eLife.36691.012
Figure 3—video 1
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Representative video demonstrating tremulousness, kyphosis, and hyperactivity in ChAT-CKO mice, as compared to controls.
https://doi.org/10.7554/eLife.36691.013
Figure 3—video 2
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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.

https://doi.org/10.7554/eLife.36691.014

Tables

Table 1
Behavioral properties of Dlx-CKO and ChAT-CKO mice.
https://doi.org/10.7554/eLife.36691.015
Motor functionDlx-CKOChAT-CKO
Pappas et al., 2015 eLife 4:e08352present manuscript
Tail suspensionTrunk twistingTrunk twisting
Forelimb clasping-
Hindlimb clasping-
-Tremulousness
Open fieldHyperactivityHypoactivity
RotarodNormalNormal
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.

https://doi.org/10.7554/eLife.36691.016
Cre expressionCell death vulnerability
Cholinergic populationDlx-CreChAT-CreDlx-CreChAT-Cre
Dorsolateral striatum
(including dorsal caudate
putamen)		ConfirmedConfirmedSevereSevere
Dorsomedial striatum
(including ventral caudate
putamen)		ConfirmedConfirmedMildSpared
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 …

https://doi.org/10.7554/eLife.36691.017
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
Gene
(Mus musculus)		Tor1aNANCBI Gene: 30931;
MGI:1353568		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
AntibodyCholine
Acetyltransferase		Millipore AB144PRRID: AB_20797511:100
AntibodyP75 Neurotrophin
Receptor		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
Antibodyanti-mouseThermoFisher
A-31571		RRID: AB_1625421:800
Antibodyanti-rabbitThermoFisher
A-21206		RRID: AB_25357921:800
Antibodyanti-rabbitThermoFisher
A-31572		RRID: AB_1625431:800
Antibodyanti-goatThermoFisher
A-21432		RRID: AB_25358531:800
Antibodyanti-goatJackson
Immunoresearch
705-065-003		RRID: AB_23403961:800
Commercial assay
or kit		ABC HRP Kit
(Standard)		Vector LaboratoriesPk-6100Vectastain elite
ABC kit
Table 4
Antibodies used for immunohistochemistry.
https://doi.org/10.7554/eLife.36691.018
LevelAntigenHostConjugatedDilutionSource
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.
https://doi.org/10.7554/eLife.36691.019
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

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.36691.020

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