Deletion of the endogenous TrkB.T1 receptor isoform restores the number of hippocampal CA1 parvalbumin-positive neurons and rescues long-term potentiation in pre-symptomatic mSOD1(G93A) ALS mice

Abstract

Amyotrophic lateral sclerosis (ALS) causes rapidly progressive paralysis and death within 5 years from diagnosis due to degeneration of the motor circuits. However, a significant population of ALS patients also shows cognitive impairments and progressive hippocampal pathology. Likewise, the mutant SOD1(G93A) mouse model of ALS (mSOD1), in addition to loss of spinal motor neurons, displays altered spatial behavior and hippocampal abnormalities including loss of parvalbumin-positive interneurons (PVi) and enhanced long-term potentiation (LTP). However, the cellular and molecular mechanisms underlying these morpho-functional features are not well understood. Since removal of TrkB.T1, a receptor isoform of the brain-derived neurotrophic factor, can partially rescue the phenotype of the mSOD1 mice, here we tested whether removal of TrkB.T1 can normalize the number of PVi and the LTP in this model. Stereological analysis of hippocampal PVi in control, TrkB.T1^-/-, mSOD1, and mSOD1 mice deficient for TrkB.T1 (mSOD1/T1^-/-) showed that deletion of TrkB.T1 restored the number of PVi to physiological level in the mSOD1 hippocampus. The rescue of PVi neuron number is paralleled by a normalization of high-frequency stimulation-induced LTP in the pre-symptomatic mSOD1/T1^-/- mice. Our experiments identified TrkB.T1 as a cellular player involved in the homeostasis of parvalbumin expressing interneurons and, in the context of murine ALS, show that TrkB.T1 is involved in the mechanism underlying structural and functional hippocampal degeneration. These findings have potential implications for hippocampal degeneration and cognitive impairments reported in ALS patients at early stages of the disease.

Keywords: Amyotrophic lateral sclerosis; Hippocampus; Long-term potentiation; Parvalbumin-positive interneurons; SOD1(G93A) mouse; TrkB.

Fig. 1.

Molecular structure of TrkB isoforms and morphofunctional components investigated in the present work. A. Schematic representation of the molecular structure of TrkB isoforms in the lipid membrane (full length = FL, truncated isoform T1). Extracellularly, the TrkB receptors are formed by a combination of Cysteine Rich Repeats, Leucine Rich Repeats and Immunoglobulin domains that is identical across isoforms. Intracellularly, the tyrosine kinase domain (TKD) is unique to the FL isoform. In the presence of brain-derived neurotrophic factor (BDNF), TrkB·FL homodimerization leads to receptor autophosphorylation at the TKD and downstream signaling (e.g., via the phosphorylation of PLC, PI3K, MAPK) to induce cell proliferation, survival/death, differentiation as well as plasticity. The TrkB.T1 isoform contains a short intracellular domain and lacks the TKD. In the presence of BDNF, T1 forms a heterotimer with FL, to exert its dominant negative inhibition (interrupted line in the figure) of FL-mediated signaling. In astrocytes, T1 is the only expressed isoform, where it controls the sequestration of BDNF. Note that while FL and T1 are the main isoforms, other isoforms exist, i.e., T2 e SHC (not shown).? = unknown intracellular signaling. B, C, D. Morphofunctional components investigated in the present study: we focus our neuroanatomical analysis on a subpopulation of hippocampal inhibitory neurons that express parvalbumin (PV), the PV+ interneurons (PVi). B. Schematics of the charts PVi; our structural approach on PVi is complemented with electrophysiological investigation (C, D) aimed at comparing synaptic plasticity at the Schaffer’s collateral, via classic LTP experiments.

Fig. 2.

The topographic distribution and gross morphology of hippocampal PVi in wt and mSOD1 mice are not influenced by TrkB.T1. Histological sections of the hippocampus illustrate the topology of PVi (20×) for representative wt (A), mSOD1 (B), T1^−/− (C), mSOD1/T1^−/− mice (D). Distribution of PVi in the different subfield is preserved across genotypes. Two insets, one for CA1 and one for CA3 (40×), display morphological features of PVi for wt (A1–A2), mSOD1 (B1–B2), T1^−/− (C1–C2), mSOD1/T1^−/− mice (D1–D2). Note how the neuronal morphology (insets) appears comparable among genotypes. In the CA1–3 subfields, PVi are mostly distributed in the stratum pyramidale, while in the DG they are found mainly in the granular layer. Both scale bars: 100 μm (large scale for insets).

Fig. 3.

TrkB.T1 shrinks the population of hippocampal PVi in wt and in mSOD1 mice. Stereological estimations of hippocampal PVi populations are shown, for each subfield, in graphs for wt (n = 6), mSOD1(n = 5), T1^−/− (n = 6), mSOD1/T1^−/− (n = 6) mice. For each graph, the ordinate indicates the PVi number. The number of PVi is comparable among groups in DG and CA2. T1 deletion results in an increase of PVi number in CA3. In mSOD1 mice both the PVi populations of CA1 and CA3 are found to be reduced compared to wt animals. Knocking out T1 in mSOD1 mice completely restores PVi population in CA1; in CA3, PVi number increases with respect to mSOD1 mice, albeit below the level of controls. Asterisks denote statistical significant differences (* = p < 0.05; *** = p < 0.01).

Fig. 4.

TrkB.T1 enhances LTP in presymptomatic mSOD1 mice. Representative fEPSP recordings from wt (A), mSOD (B) and mSOD1/T1^−/− (C) animals. For each case, the traces are obtained before (gray) and 50 min (green) after the high-frequency stimulation (HFS) conditioning protocol (2 times at 100 ms, 100 Hz trains). The mSOD1 mice (n = 8 slices) exhibit enhanced levels - with respect to wt mice (n = 9 slices) - of long-term potentiation (LTP) 50 min following HFS; at the same time point, mSOD1/T1^−/− mice (n = 8 slices) display levels of LTP resembling those of wt mice (D). Following HFS, the potentiation of fEPSP is higher in mSOD1 mice compared to wt mice; the post-HFS fEPSP is comparable between wt mice and mSOD1/T1^−/− mice (E). Asterisks denote statistical significant differences (* = p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)