Differentiation defects in primary motoneurons from a SMARD1 mouse model that are insensitive to treatment with low dose PEGylated IGF1

Abstract

Muscle atrophy and diaphragmatic palsy are the clinical characteristics of spinal muscular atrophy with respiratory distress type 1 (SMARD1), and are well represented in the neuromuscular degeneration (Nmd(2J) ) mouse, modeling the juvenile form of SMARD1. Both in humans and mice mutations in the IGHMBP2 gene lead to motoneuron degeneration. We could previously demonstrate that treatment with a polyethylene glycol-coupled variant of IGF1 (PEG-IGF1) improves motor functions accompanied by reduced fiber degeneration in the gastrocnemius muscle and the diaphragm, but has no beneficial effect on motoneuron survival. These data raised the question which cell autonomous disease mechanisms contribute to dysfunction and loss of Ighmbp2-deficient motoneurons. An analysis of primary Ighmbp2-deficient motoneurons exhibited differentiation deficits such as reduced spontaneous Ca(2+) transients and altered axon elongation, which was not compensated by PEG-IGF1. This points to an IGF1 independent mechanism of motoneuron degeneration that deserves treatment approaches in addition to IGF1.

Keywords: Cav2.2; IGF1; Ighmbp2; SMARD1; motoneurons.

Figure 1. Disturbed calcium homeostasis at distal axons and altered Ca_v2.2 accumulation at growth cones of Ighmbp2-deficient motoneurons on the synapse-specific laminin-221. (A) Spontaneous Ca²⁺ transients in isolated Ighmbp2-deficient motoneurons were markedly reduced in the growth cone compartment (Controls 0.30 ± 0.06 min⁻¹, n = 22, vs. Ighmbp2 def. 0.09 ± 0.02 min⁻¹, n = 73; U = 487, p** = 0.0047). The Ca²⁺ transient frequency in the cell body of Ighmbp2-deficient motoneurons was not affected (Controls 0.56 ± 0.10 min⁻¹, n = 116, vs. Ighmbp2 def. 0.62 ± 0.11 min⁻¹, n = 45; U = 2,152, P = 0.08). (B) The quantitative analysis of Ca_v2.2 presence in Ighmbp2-deficient growth cones revealed a significant reduction (Controls 119.3 ± 3.5 a.u., n = 92, vs. Ighmbp2 def. 77.1 ± 2.6 a.u., n = 106; U = 1,622, p*** < 0.001). In (B’, B”’) a decreased Ca_v2.2 accumulation in the protrusions of Ighmbp2-deficient growth cones is displayed, co-labeled with the active zone marker Bassoon (B”, B””). Analysis of Ca_v1.3 channels showed neither an altered distribution (C’, C”) nor reduced levels (C) in Ighmbp2-deficient motoneurons (Controls 22.7 ± 0.8 a.u., n = 76, vs. Ighmbp2 def. 21.7 ± 0.7 a.u., n = 81; U = 3,000, P = 0.78). (D) No difference in signal intensity of synaptophysin was detected between Ighmbp2-deficient and control motoneurons (Controls 25.9 ± 1.6 a.u., n = 90, vs. Ighmbp2 def. 27.3 ± 1.4 a.u., n = 90; U = 3,552, P = 0.15). Synaptophysin was evenly distributed throughout the growth cone compartment and exhibited no difference regarding localization between Ighmbp2-deficient (D”’, D””) and control motoneurons (D’, D”). Single values were obtained from at least three different experiments. Statistical analysis was performed using Mann-Whitney t test. Bars represent mean ± SEM, significance is indicated by stars (p** < 0.01, p*** < 0.001, non-significance is indicated by n.s.).

Figure 2. Low doses of PEG-IGF1 do not compensate for the axon elongation defect in Ighmbp2-deficient motoneurons on the synapse-specific laminin-221. In (A-C) the quantitative analyses of axon length, dendrite length, and growth cone size of Ighmbp2-deficient and control motoneurons are shown. (A) Isolated Ighmbp2-deficient motoneurons exhibited an altered axonal processing on laminin-221 (Controls 605.7 ± 13.4 µm, n = 585, vs. Ighmbp2 def. 780.9 ± 19.8 µm, n = 492; U = 104,000, p*** < 0.001). Representative images are shown in (A’, A”). (B) The dendrite length of Ighmbp2-deficient motoneurons was comparable to the control situation (Controls 29.5 ± 0.8 µm, n = 292, vs. Ighmbp2 def. 28.4 ± 0.8 µm, n = 206; U = 30,010, P = 0.97). In (C) a markedly growth cone size reduction is depicted (Controls 67.0 ± 5.2 µm², n = 78, vs. Ighmbp2 def. 44.7 ± 2.4 µm², n = 102; U = 2,739, p*** = 0.0004). (D) The β-actin distribution and localization was disturbed in growth cones of Ighmbp2-deficient motoneurons (Controls 84.0 ± 3.0 a.u., n = 105, vs. Ighmbp2 def. 64.0 ± 2.1 a.u., n = 107; U = 3,486, p*** < 0.001). Representative images are shown in (D’, D”). In (E) the quantification of axon lengths from control and Ighmbp2-deficient motoneurons in the presence of CNTF (C), BDNF (B) (10 ng/ml, each), and PEG-IGF1 (I) (1 ng/ml), and PEG-IGF1 (I) (1 ng/ml) alone is depicted (C+B+I: Controls 558.9 ± 14.6 µm, n = 375, vs. Ighmbp2 def. 711.7 ± 18.8 µm, n = 402; U = 55,050, p*** < 0.001. I: Controls 498.4 ± 13.6 µm, n = 301, vs. Ighmbp2 def. 577.7 ± 22.7 µm, n = 190; U = 25,090, p* = 0.02). Single values were obtained from at least three different experiments. Statistical analysis was performed using Mann-Whitney t test. Bars represent mean ± SEM, significance is indicated by stars (p* < 0.05, p*** < 0.001, non-significance is indicated by n.s.).