Cytoskeletal changes during development and aging in the cortex of neurofilament light protein knockout mice

Abstract

The neurofilament light (NFL) subunit is considered as an obligate subunit polymer for neuronal intermediate filaments comprising the neurofilament (NF) triplet proteins. We examined cytoskeletal protein levels in the cerebral cortex of NFL knockout (KO) mice at postnatal day 4 (P4), 5 months, and 12 months of age compared with age-matched wild-type (WT) mice of a similar genetic background (C57BL/6). The absence of NFL protein resulted in a significant reduction of phosphorylated and dephosphorylated NFs (NF-P, NF-DP), the medium NF subunit (NFM), and the intermediate filament α-internexin (INT) at P4. At 5 months, NF-DP, NFM, and INT remained significantly lower in knockouts. At 12 months, NF-P was again significantly decreased, and INT significantly increased, in KOs compared with wild type. In addition, protein levels of class III neuron-specific β-tubulin and microtubule-associated protein 2 were significantly increased in NFL KO mice at P4, 5 months, and 12 months, whereas β-actin levels were significantly decreased at P4. Immunocytochemical studies demonstrated that NF-DP accumulated abnormally in the perikarya of cortical neurons by 5 months of age in NFL KO mice. Neurons that lacked NF triplet proteins, such as calretinin-immunolabeled nonpyramidal cells, showed no alterations in density or cytoarchitectural distribution in NFL KO mice at 5 months relative to WT mice, although calretinin protein levels were decreased significantly after 12 months in NFL KO mice. These findings suggest that a lack of NFL protein alters the expression of cytoskeletal proteins and disrupts other NF subunits, causing intracellular aggregation but not gross structural changes in cortical neurons or cytoarchitecture. The data also indicate that changes in expression of other cytoskeletal proteins may compensate for decreased NFs.

Changes in TDP-43 expression in development, aging, and in the neurofilament light protein knockout mouse

“…NF-L KO mice were maintained on a C57BL/6 background as a homozygous colony (Liu et al, 2013;Zhu et al, 1997). Western blotting was used to confirm the absence of NF-L protein in the NF-L KO mice (data not shown).…”

“…Sections processed without the primary antibody were used as controls. Fluorescence-labeled sections were viewed with a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Oberkochen, Germany) and images acquired as described previously (Liu et al, 2013).…”

“…We and others have shown that a lack of NF-L protein alters the expression of cytoskeletal proteins and disrupts other NF subunits, including decreased expression of NF-M and NF-H (Liu et al, 2013), causing perikaryal accumulation of NF proteins (Liu et al, 2013;Zhu et al, 1997), thus mimicking to some degree the cytoskeletal changes described in ALS (Wong et al, 2000). A recent study described that Rho guanine nucleotide exchange factor is an NF-L mRNA destabilizing factor that forms cytoplasmic inclusions in ALS, suggesting that altered motor neuron RNA metabolism can explain the development of neurofilamentous aggregates, observed in ALS pathology (Droppelmann et al, 2013).…”

“…Protein levels of NF-L and TDP-43 in the brain were examined across a range of embryonic and postnatal ages in wild-type (WT) mice. We also investigated whether the absence of NF-L expression could drive alterations to TDP-43 expression, localization, or pathology by using mice in which the NF-L gene has been knocked out (NF-L KO), which results in a substantial reduction of intermediate filaments in neurons (Liu et al, 2013;Zhu et al, 1997). To examine whether these changes alter the levels or location of TDP-43 expression, the protein levels of TDP-43, including phosphorylated S403/404 and S409/410 forms, were investigated in the brain and spinal cord of young and aged NF-L KO mice, relative to age-matched WT mice from the same genetic background (C57BL/6).…”

Adolescent maturation of the prefrontal cortex: Role of stress and sex in shaping adult risk for compromise

“…Variation in the development and stability of cytoskeletal proteins could lead to major changes in adulthood yet have not been explicitly studied either between the sexes or within the scope of stressors during adolescent development of the mPFC. The highly dynamic nature of cytoskeletal proteins allow growth and plasticity of neural cells for fast adaptation to novel scenarios, yet also introduces an additional level of susceptibility of neural cells to environmental insult, such as psychosocial stressors . For instance, psychosocial stress and increased circulating corticosterone levels alter actin microfilament, neurofilament and microtubule stability, as well as alterations in dendritic morphology via TrkB and β‐integrin mediated signaling in adolescence .…”