Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

Abstract

Dynactin is a complex motor protein involved in the retrograde axonal transport disturbances of which may lead to amyotrophic lateral sclerosis (ALS). Mice with hSOD1G93A mutation develop ALS-like symptoms and are used as a model for the disease studies. Similar symptoms demonstrate Cra1 mice, with Dync1h1 mutation. Dynactin heavy (DCTN1) and light (DCTN3) subunits were studied in the CNS of humans with sporadic ALS (SALS), mice with hSOD1G93A (SOD1/+), Dync1h1 (Cra1/+), and double (Cra1/SOD1) mutation at presymptomatic and symptomatic stages. In SALS subjects, in contrast to control cases, expression of DCTN1-mRNA but not DCTN3-mRNA in the motor cortex was higher than in the sensory cortex. However, the mean levels of DCTN1-mRNA and protein were lower in both SALS cortexes and in the spinal cord than in control structures. DCTN3 was unchanged in brain cortexes but decreased in the spinal cord on both mRNA and protein levels. In all SALS tissues immunohistochemical analyses revealed degeneration and loss of neuronal cells, and poor expression of dynactin subunits. In SOD1/+ mice both subunits expression was significantly lower in the frontal cortex, spinal cord and hippocampus than in wild-type controls, especially at presymptomatic stage. Fewer changes occurred in Cra1/SOD1 and Cra1/+ mice.It can be concluded that in sporadic and SOD1-related ALS the impairment of axonal retrograde transport may be due to dynactin subunits deficiency and subsequent disturbances of the whole dynein/dynactin complex structure and function. The Dync1h1 mutation itself has slight negative effect on dynactin expression and it alleviates the changes caused by SOD1G93A mutation.

Fig. 1

Expression of dynactin DCTN1 and DCTN3 mRNA in the CNS of SALS and control cases. The expression was studied by real-time qPCR, as described in the Material and method section. The results were quantified as the ratio of studied dynactin (DCTN1 or DCTN3) expression to the expression of housekeeping genes (B2M and GusB; ΔCt method). No. 1–5, cases with SALS; a–e, control cases. Black bars motor cortex; striped bars sensory cortex

Fig. 2

Expression of dynactin DCTN1 and DCTN3 protein in the CNS of SALS and control cases. The expression was studied by Western blotting, as indicated in the Material and method section. Comparable amounts of protein (20 μg for DCTN1 and 40 μg for DCTN3) from representative control (no D) and SALS (no 4) brains were run in each lane. A; DCTN1: line 1 control motor cortex (optical density: OD 651), line 2 control sensory cortex (OD 847), line 3 SALS motor cortex (OD 439), line 4 SALS sensory cortex (OD 519), line 5 control spinal cord (OD 940), line 6 SALS spinal cord (OD 627). b DCTN3: line 1 control motor cortex (OD 211), line 2 control sensory cortex (OD 238), line 3 SALS motor cortex (OD 210), line 4 SALS sensory cortex (OD 290), line 5 control spinal cord (OD 180), line 6 SALS spinal cord (OD 92)

Fig. 3

Representative immunohistochemistry for dynactin DCTN1 on sections from SALS and control human cases. a Immunopossitive neurons and their processes in SALS motor cortex and visible moderate loss of neuronal cells; b positive immunoreactivity in SALS neurons and axons in the sensory cortex; c moderate immunoexpression in pericaria and axons of the preserved SALS anterior horn motoneurons with features of degeneration; d pronounced immunoreactivity of control neuronal processes and pericaria in the motor cortex; e positive immune reaction of different intensity in control axons and neuronal pericaria in the sensory cortex; f very strong immunoreactivity of processes and motoneuron pericaria in control spinal cord anterior horn. Bars 100 μm each

Fig. 4

Representative immunohistochemistry for dynactin DCTN3 on sections from SALS and control human cases. a Weakly immunopositive single neuron in SALS motor cortex (arrow) and visible loss of neurons; b mostly negative immune reaction in SALS neurons and axons (arrow) in the sensory cortex; c very poor or absent immunoexpression in the preserved motoneurons with features of degeneration in the anterior horn of SALS spinal cord; d weak immunolabel in pericaria of neurons and pronounced in their processes in the control motor cortex; e strong immunoreactivity of axons and poor of neuronal pericaria in the control sensory cortex; f weak immunoexpression in control pericaria of anterior horn motoneurons and mild in axons (arrows). Bars 100 μm

Fig. 5

Expression of dynactin Dctn1 or Dctn3 mRNA in the CNS of transgenic mice. The expression was determined by RT-PCR and expressed as the ratio of the optical density (OD) value of Dctn1 or Dctn3 to the optical density of S12 protein RNA, as indicated in the Material and Method section. Filled diamond dashed lines wild-type controls (+/+); filled triangles Cra1/+ mice; filled circles SOD1/+ mice; filled squares Cra1/SOD1 hybrides