Análisis del Proyecto

Interneurons are essential components of the cortical motor neuron network and may be central to network failure. Every segment of a pyramidal neuron, such as soma, dendritic branches and spines, and the initial axonal segment, receives dense GABAergic synaptic innervation [see reviews (Jones 1993, Buzsaki et al 2004)]. In ALS, clinical imaging studies suggest brain plasticity in motor networks is correlated with disease progression (Poujois et al 2013), and the extent of inhibitory alteration in the motor cortex is associated with increased excitability of the motor cortex (Menon et al 2014, Geevasinga et al 2015, Shibuya et al 2016). Hence, this study was interested in determining if such a correlation may be found in the interneuron and pyramidal pathology in the region.

The results of this study show a correlation between differential loss of CR-interneurons in the primary motor cortex and pyramidal neuron pathology. That is, in the primary motor cortex of ALS cases, the selective loss of CR-interneurons was associated with pyramidal neuron loss, measured by decreased density of layer V pyramidal neurons. Intriguingly, the converse was also shown with preservation of CR-interneurons associated with a lesser extent of pyramidal neuron pathology. This suggests that interneuron pathology and corticomotoneuronal pathology may be coupled in the ALS motor cortex. However, as CB-interneuron pathology is not associated with pyramidal neuron pathology, this indicates vulnerable networks may be associated with specific interneuron populations, such as the CR-interneurons.

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From a functional view point, when looking at other diseases with demonstrated reductions in CR- interneurons, it is most interesting to find that in the epileptic human hippocampus the number of CR-cells lost also correlated with the severity of principal cell loss in the region (Toth et al 2010). This common neurological condition is by definition due to abnormal excessive or synchronous neuronal activity in the brain (Fisher et al 2005), which when comparing evidence for hyperexcitability in the motor cortex in ALS draws certain parallels. Thus, when considered in the context of ALS, this may suggest a pathogenic feedback mechanism whereby the loss of CR cells translates to a loss of principal cells, or vice verse. Additionally, it may also indicate that reduced CR populations are involved in a loss of circuitry control, contributing to the hyperexcitability observed in ALS, and epilepsy.

While it remains to be determined if CR-populations drive corticomotoneuronal degeneration, or respond to altered network feedback due to corticomotoneuronal degeneration, the differential nature of CR pathology in patients is not surprising. Previous literature has established that the extent of pyramidal neuron pathology in layer V of the motor cortex varies between patients (Nihei et al 1993, Maekawa et al 2004), as also observed in this study. Therefore, it was quite unexpected that there was an overall significant increase in NPY interneurons throughout the entire motor cortex of all ALS cases. However, in line with the clustering of ALS cases based on CR-interneuron pathology, it was an interesting observation to find that cases with the least amount of pyramidal and CR- pathology had the greatest increase in NPY cell density relative to controls. Indeed, this study shows a correlation of NPY-pathology with both pyramidal pathology and CR-pathology. This suggests that while CR-interneuron pathology may be detrimental in the motor cortex, increased NPY may be associated with a reduced extent of cortical damage. However, this correlation was only found in ALS cases, not controls, suggesting this may be a compensatory mechanism specifically related to an altered network in the ALS motor cortex. This may be supported by the apparent marked increase in NPY-immunoreactive fibres identified to varying degrees in all ALS motor cortices studied.

When considering the distinct morphological appearance of NPY-immunoreactive fibres in the ALS cortex, again similar parallels are observed with key-disease affected regions in the epileptic brain. Previous studies have shown an increase in the cell density and length of NPY immunoreactive fibres in hippocampal subfields with sclerosis in the epileptic human hippocampus (De Lanerolle et al 1989, Mathern et al 1995, Furtinger et al 2001), which mirror the NPY pathology detected in the present study. The striking similarity of this pathology is best demonstrated when directly comparing photomicrographs from works of Furtinger and colleagues (Furtinger et al 2001) with images from the present study (Figure 3.6).

Figure 3.6. NPY-immunoreactive fibres in the epileptic human hippocampus parallel NPY-pathology demonstrated in the ALS motor cortex.

a, Widespread NPY immunoreactivity is shown in a patient with hippocampal sclerosis as demonstrated by Furtinger and colleagues in 2001. b, Striking similarities are demonstrated in the NPY-immunoreactive fibres found in the ALS motor cortex in this study. High magnification images in both show the unusual pattern of NPY process labelling in both disease-associated regions. Scale bar in (a-b) 50µm.

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While the mechanism responsible for NPY involvement in ALS is not yet known, it is tempting to speculate that this may again represent a mechanism related to increased excitability. Regarding modes of NPY expression, in normal neurological conditions NPY is contained and released by GABAergic interneurons, however in regions of pathogenically enhanced excitability it can be found upregulated in interneurons and aberrantly expression in non-neuronal populations (Marksteiner et al 1990, Rizzi et al 1993, Gruber et al 1994). This has been typically demonstrated in the hippocampus of mouse and rat strains that are susceptible to seizures, with NPY transiently upregulated in interneurons and granule cells/mossy fibres of the dentate gyrus from the pre-convulsive stage in animals (Schwarzer et al 1996, Vezzani et al 1996). Thus, it is possible that NPY-fibre immunoreactivity in this study may represent upregulation of the neuropeptide on either GABAergic or glutamatergic processes. While we confirm that CR-interneuron involvement is restricted to aspiny inhibitory SMI32 negative populations, future studies should also conduct co-localisation for equal assessment of NPY labelling. Hence while we cannot rule out the upregulation of NPY on pyramidal populations, it is important to note that irrespective of the mode of delivery, administration of NPY has been found to potently suppress epileptic activity in hippocampal slices from epilepsy patients (Patrylo et al 1999). Therefore, the demonstrated alteration of NPY reported here should remain of high interest in the context of cortical hyperexcitability demonstrated in the motor cortex of ALS patients.