Knockout of Amyloid ß Protein Precursor (APP) Expression Alters Synaptogenesis, Neurite Branching and Axonal Morphology of Hippocampal Neurons.

The function of the ß-A4 amyloid protein precursor (APP) of Alzheimer's disease (AD) remains unclear. APP has a number of putative roles in neuronal differentiation, survival, synaptogenesis and cell adhesion. In this study, we examined the development of axons, dendrites and synapses in cultures of hippocampus neutrons derived from APP knockout (KO) mice. We report that loss of APP function reduces the branching of cultured hippocampal neurons, resulting in reduced synapse formation. Using a compartmentalised culture approach, we found reduced axonal outgrowth in cultured hippocampal neurons and we also identified abnormal growth characteristics of isolated hippocampal neuron axons. Although APP has previously been suggested to play an important role in promoting cell adhesion, we surprisingly found that APPKO hippocampal neurons adhered more strongly to a poly-L-lysine substrate and their neurites displayed an increased density of focal adhesion puncta. The findings suggest that the function of APP has an important role in both dendritic and axonal growth and that endogenous APP may regulate substrate adhesion of hippocampal neurons. The results may explain neuronal and synaptic morphological abnormalities in APPKO mice and the presence of abnormal APP expression in dystrophic neurites around amyloid deposits in AD.

Amyloid ß precursor protein regulates neuron survival and maturation in the adult mouse brain.

The amyloid-ß precursor protein (APP) is a transmembrane protein that is widely expressed within the central nervous system (CNS). While the pathogenic dysfunction of this protein has been extensively studied in the context of Alzheimer's disease, its normal function is poorly understood, and reports have often appeared contradictory. In this study we have examined the role of APP in regulating neurogenesis in the adult mouse brain by comparing neural stem cell proliferation, as well as new neuron number and morphology between APP knockout mice and C57bl6 controls. Short-term EdU administration revealed that the number of proliferating EdU+ neural progenitor cells and the number of PSA-NCAM+ neuroblasts produced in the SVZ and dentate gyrus were not affected by the life-long absence of APP. However, by labelling newborn cells with EdU and then following their fate over-time, we determined that ~48% more newly generated EdU+ NeuN+ neurons accumulated in the granule cell layer of the olfactory bulb and ~57% more in the dentate gyrus of young adult APP knockout mice relative to C57bl6 controls. Furthermore, proportionally fewer of the adult-born olfactory bulb granule neurons were calretinin+. To determine whether APP was having an effect on neuronal maturation, we administered tamoxifen to young adult Nestin-CreERT2::Rosa26-YFP and Nestin-CreERT2::Rosa26-YFP::APP-knockout mice, fluorescently labelling ~80% of newborn (EdU+) NeuN+ dentate granule neurons formed between P75 and P105. Our analysis of their morphology revealed that neurons added to the hippocampus of APP knockout mice have shorter dendritic arbors and only half the number of branch points as those generated in C57bl6 mice. We conclude that APP reduces the survival of newborn neurons in the olfactory bulb and hippocampus, but that it does not influence all neuronal subtypes equally. Additionally, APP influences dentate granule neuron maturation, acting as a robust regulator of dendritic extension and arborisation.

Axonal degeneration, distal collateral branching and neuromuscular junction architecture alterations occur prior to symptom onset in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Degeneration of the distal axon and neuromuscular junction (NMJ) is considered a key and early feature of the pathology that accompanies motor neuron loss in people with amyotrophic lateral sclerosis (ALS). The mutant SOD1(G93A) mouse replicates many features of the disease, however the sequence of events resulting in degeneration of the neuromuscular circuitry remains unknown. Furthermore, despite widespread degenerative neuronal pathology throughout the spinal cord in this model, hindlimb motor function is lost before forelimb function. We investigated axons and NMJs in the hindlimb (gastrocnemius) and forelimb (extensor) muscles in the high copy number mutant SOD1(G93A)xYFP (yellow fluorescent protein) mouse. We found that distal axonal and NMJ alterations were present prior to previously reported functional symptom onset in this strain. Indeed, increased branch complexity as well as colocalisation between pre- and post-synaptic markers indicated widespread early axonal and NMJ alterations in the hindlimb. Immunohistochemical analysis demonstrated that the colocalisation of the scaffolding proteins nestin, LRP-4, dystrophin and rapsyn were diminished before post-synaptic receptors in the gastrocnemius, and the degree of loss differed between proteins. Analysis of the forelimb muscle revealed axonal and NMJ degeneration at a late, post symptomatic stage, as well as novel differences in NMJ morphology, with reduced complexity. Furthermore, post-synaptic scaffolding proteins were preserved in the forelimb compared with the hindlimb. Analysis of protein levels indicated an increase in LRP-4, dystrophin and rapsyn in post symptomatic skeletal muscle that may suggest ongoing attempts at repair. This study indicates that axonal and NMJ degeneration in the SOD1 model of ALS is a complex and evolving sequence of events. We provide evidence that YFP can detect morphological and plastic alterations in the SOD1(G93A) mouse, and that the pre- and post-synaptic integrity of the NMJ plays an important role in the pathogenic mechanisms of ALS.

Do Microglia Default on Network Maintenance in Alzheimer's Disease?

Although the cause of Alzheimer's disease (AD) remains unknown, a number of new findings suggest that the immune system may play a critical role in the early stages of the disease. Genome-wide association studies have identified a wide array of risk-associated genes for AD, many of which are associated with abnormal functioning of immune cells. Microglia are the brain's immune cells. They play an important role in maintaining the brain's extracellular environment, including clearance of aggregated proteins such as amyloid-ß (Aß). Recent studies suggest that microglia play a more active role in the brain than initially considered. Specifically, microglia provide trophic support to neurons and also regulate synapses. Microglial regulation of neuronal activity may have important consequences for AD. In this article we review the function of microglia in AD and examine the possible relationship between microglial dysfunction and network abnormalities, which occur very early in disease pathogenesis.

Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUS-associated ALS.

Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER-Golgi transport is a novel therapeutic target in ALS.

Identifying the primary site of pathogenesis in amyotrophic lateral sclerosis - vulnerability of lower motor neurons to proximal excitotoxicity.

There is a desperate need for targeted therapeutic interventions that slow the progression of amyotrophic lateral sclerosis (ALS). ALS is a disorder with heterogeneous onset, which then leads to common final pathways involving multiple neuronal compartments that span both the central and peripheral nervous system. It is believed that excitotoxic mechanisms might play an important role in motor neuron death in ALS. However, little is known about the mechanisms by which excitotoxicity might lead to the neuromuscular junction degeneration that characterizes ALS, or about the site at which this excitotoxic cascade is initiated. Using a novel compartmentalised model of site-specific excitotoxin exposure in lower motor neurons in vitro, we found that spinal motor neurons are vulnerable to somatodendritic, but not axonal, excitotoxin exposure. Thus, we developed a model of somatodendritic excitotoxicity in vivo using osmotic mini pumps in Thy-1-YFP mice. We demonstrated that in vivo cell body excitotoxin exposure leads to significant motor neuron death and neuromuscular junction (NMJ) retraction. Using confocal real-time live imaging of the gastrocnemius muscle, we found that NMJ remodelling preceded excitotoxin-induced NMJ degeneration. These findings suggest that excitotoxicity in the spinal cord of individuals with ALS might result in a die-forward mechanism of motor neuron death from the cell body outward, leading to initial distal plasticity, followed by subsequent pathology and degeneration.

Microfluidic primary culture model of the lower motor neuron-neuromuscular junction circuit.

Modelling the complex process of neuromuscular signalling is key to understanding not only normal circuit function but also importantly the mechanisms underpinning a range of degenerative diseases. We describe a novel in vitro model of the lower motor neuron-neuromuscular junction circuit, incorporating primary spinal motor neurons, supporting glia and skeletal muscle. This culture model is designed to spatially mimic the unique anatomical and cellular interactions of this circuit in compartmented microfluidic devices, such that the glial cells are located with motor neuron cell bodies in the cell body chamber and motor neuron axons extend to a distal chamber containing skeletal muscle cells whilst simultaneously allowing targeted intervention. This model is suitable for use in conjunction with a range of downstream experimental approaches and could also be modified to utilise other cellular sources including appropriate immortal cell lines, cells derived from transgenic models of disease and also patient derived stem cells.

Degeneration of axons in spinal white matter in G93A mSOD1 mouse characterized by NFL and α-internexin immunoreactivity.

Axonal degeneration is a prominent feature of amyotrophic lateral sclerosis (ALS) both in lower motor nerves as well as descending white matter axons in the spinal cord of human patients. Although the pathology of lower motor axonal degeneration has been described in both human ALS and related transgenic animal models, few studies have examined the pathological features of descending axon degeneration, particularly in mouse models of ALS. We have examined the degeneration of white matter tracts in the G93A mutant superoxide dismutase-1 (mSOD1+) mouse spinal cord white matter from 12 weeks of age to end-stage disease. In a G93A mSOD1 mouse model where green fluorescent protein was expressed in neurons (mSOD1+/GFP+), degeneration of white matter tracts was present from the ventral to dorsolateral funiculi. This pattern of axonal pathology occurred from 16 weeks of age. However, the dorsal funiculus, the site of the major corticospinal tract in mice, showed relatively less degeneration. Immunohistochemical analysis demonstrated that the neurofilament light chain (NFL) and neuronal intermediate filament protein alpha-internexin accumulated in axon swellings in the spinal white matter. Increased levels of alpha-internexin protein, in mSOD1+ mouse spinal cord tissue, were demonstrated by Western blotting. In contrast, degenerating axons did not show obvious accumulations of neurofilament medium and heavy chain proteins (NFM and NFH). These data suggest that white matter degeneration in this mouse model of ALS is widespread and involves a specific molecular signature, particularly the accumulation of NFL and alpha-internexin proteins.