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Cornelia Ringer, Eberhard Weihe, Burkhard Schütz, SOD1G93A Mutant Mice Develop a Neuroinflammation-Independent Dendropathy in Excitatory Neuronal Subsets of the Olfactory Bulb and Retina, Journal of Neuropathology & Experimental Neurology, Volume 76, Issue 9, September 2017, Pages 769–778, https://doi.org/10.1093/jnen/nlx057
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Abstract
Nonmotor neuron-related pathology is a feature of amyotrophic lateral sclerosis (ALS), both in patients and in animal models. There is emerging evidence that sensory systems (olfaction and vision) are affected in humans. Here, we asked whether such sensory neuropathology is recapitulated in the superoxide dismutase 1 (SOD1G93A) mouse model of ALS. Neuronal vacuolization in olfaction and vision pathways was assessed in tissue sections from presymptomatic and symptomatic disease stages, and compared to wild type. In both, the olfactory bulb and retina, vacuolization started around postnatal day 60, and vacuole sizes increased until disease end-stage. Notably, vacuolization was largely restricted to the external plexiform layer of the olfactory bulb and to the inner plexiform layer of the retina. In both layers, hSOD1-immunoreactive vacuoles localized to dendrites of excitatory neurons. Downstream olfaction and vision pathway fiber tracts and relay stations did not display obvious vacuolization. Finally, on a morphological level, there was no evidence for an activation of astrocytes and microglia in the 2 affected areas. Thus, we identified a new pathology hallmark in SOD1G93A ALS mice: a glutamatergic sensory neuron dendropathy restricted to olfactory bulb mitral cells and retinal ganglionic cells.
INTRODUCTION
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that predominantly affects upper and lower somatomotor neurons in the cortex and spinal cord. Progressive muscle weakness and paralysis typically results in death due to respiratory failure 3–5 years after diagnosis (1, 2). While 90% of the cases occur spontaneously (sporadic ALS), the remaining 10% are inherited (familial ALS). Mutations have been discovered in several genes, including superoxide dismutase 1 (SOD1), TAR DNA-binding protein-43 (TDP-43), fused in sarcoma, Optineurin, valosin-containing protein, ubiquilin 2 (UBQLN2), and chromosome 9 open reading frame 72 (C9orf72) (3).
During the last decade it became apparent that ALS represents a multisystem disorder that also displays extrapyramidal, autonomic, and cognitive pathology and symptomatology in a subset of patients (4–6). In addition, impairments in sensory systems (olfaction and vision) have been described (7–13). However, while some ALS patients showed severe olfactory impairments in odor tests, others were without symptomatology, and autopsies showed histopathology in either olfactory bulb neurons or the limbic system (12–15). On the other hand, optical coherence tomography in ALS patients in vivo revealed a thinning of select retinal layers as a constant finding (8–10).
Using the vacuolization of mitochondria as a marker for neurodegeneration (16–20), pathologies of the extrapyramidal and autonomic motor systems already have been shown to also occur in the SOD1G93A mouse model of ALS (20, 21), while sensory system pathology has not yet been investigated in detail. In our previous study researching correlations between severity of degeneration-related neuron vacuolization and the degree of astrogliosis throughout the brain of SOD1G93A mice, we identified signs of neuropathology in the olfactory bulb of end-stage animals (22). Here, we determined the localization and time course of the neuronal vacuolization in the olfactory bulb in detail and extended our investigation to downstream fiber tracts and relay areas of the olfactory pathway. In addition, the visual pathway from the retina to the primary visual cortex was included in our analysis. Overall, our study aimed to decipher as to whether human sensory system pathology is recapitulated in the SOD1G93A and thus most commonly used mouse model of ALS, a finding that would open new avenues for experimentally elucidating the molecular mechanisms that drive degeneration of certain neuron types in ALS.
MATERIALS AND METHODS
Compliance With Ethical Standards
All animal procedures were conducted in accordance with international standards on animal welfare, the European directive 2010/63/EU, and with the German Animal Protection Law under a protocol approved by the county administrative government authorities in Giessen, Germany (reg.nr. 48/2010) (22).
Mouse Strain and Tissue Processing
Transgenic mice of the strain B6SJL-TgN(SOD1(G93A)) 1Gur (The Jackson Laboratory, Bar Harbor, ME) carrying human SOD1 with the pathogenic G93A mutation (SOD1G93A) in high copy number (23) were used as mouse model for ALS. The breeding scheme, housing conditions, clinical assessments of disease progression, and selection of controls of these mice have been described previously (22).
Mice were killed by exposure to an overdose of inhaled isoflurane followed by pneumothorax. Both eyes and the brain including the olfactory bulb were dissected from 4 to 5 animals per genotype and disease stage, and immersion-fixed for 48 hours in Bouin Hollande fixative containing 4% (w/v) picric acid, 2.5% (w/v) cupric acetate, 3.7% (v/v) formaldehyde, and 1% (v/v) glacial acetic acid. Following fixation, the tissues were extensively washed in 70% isopropanol, dehydrated, cleared with xylene, and embedded in paraffin. Seven micrometer-thick sections were cut with a microtome and mounted onto silanized glass slides. Histological stains on select sections were done with Giemsa stain.
Single Immunohistochemistry
Single immunohistochemistry was performed as described previously (22). Briefly, tissue sections were deparaffinized in xylene and rehydrated through a graded series of isopropanol, including 30-minute incubation in methanol/0.3% hydrogen peroxide to block endogenous peroxidase activity. Antigen retrieval was achieved by incubation in 10 mM sodium citrate buffer (pH 6.0) at 92 °C–95 °C for 15 minutes, and nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) in 50 mM phosphate buffered saline ([PBS], pH 7.45) for 30 minutes, followed by an avidin–biotin blocking step (Avidin–Biotin Blocking Kit, Boehringer, Ingelheim, Germany) for 20 minutes each. Subsequently, primary antibodies (Table) were applied in PBS/1% BSA over night at 16 °C followed by 2 hours at 37 °C. After several washes in PBS, the sections were incubated for 45 minutes at 37 °C with species-specific biotinylated secondary antibodies (Dianova, Hamburg, Germany), diluted 1:200 in PBS/1% BSA, washed, and incubated for 30 minutes with avidin–biotin–peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Immunoreactions were then visualized by 8-minute incubation in 3,3-diaminobenzidine ([DAB], Sigma Aldrich, Deisenhofen, Germany), enhanced by the addition of 0.08% ammonium nickel sulfate (Fluka, Bucks, Switzerland). After 3–5-minute washes in distilled water, the sections were dehydrated through a graded series of isopropanol, cleared in xylene, and finally mounted under coverslips. Digital bright-field pictures were taken with a Zeiss Axio Imager M2 microscope (Zeiss, Oberkochem, Germany), equipped with a Zeiss AxioCam HRc camera and ZEN Lite Analyses software (Zeiss).
Antibody (Abbr.) . | Target PROTEIN . | Specificity . | Source . | Donor Species . | Working Dilution (BF/DF) . |
---|---|---|---|---|---|
GAD65/67 | Glutamic acid decarboxylases 65 and 67 | Inhibitory, GABAergic neurons | Abcam (ab49832) | rb | n.a./100 |
GFAP | Glial fibrillary acidic protein | Astrocytes | Progen (GP52) | gp | 5.000/n.a. |
Iba1 | Ionized calcium-binding adaptor protein 1 | Microglia | Wako Chemicals (019-19741) | rb | 500/n.a. |
MAP2 | Microtubule-associated protein 2 | Neuronal cell bodies and dendrites | Abcam (ab183830) | rb | n.a./500 |
NF200 | Neurofilament heavy chain 200kD | Neuronal cell bodies and axons | Abcam (ab8135) | rb | n.a./100 |
PGP9.5 | Protein-gene-product 9.5 | Excitatory neurons | Cedarlane (CL95101) | rb | n.a./1.000 |
SOD1 | Superoxide dismutase 1 | Human SOD1 | Santa Cruz Biotech. (sc-8637) | gt | 1.000/500 |
Antibody (Abbr.) . | Target PROTEIN . | Specificity . | Source . | Donor Species . | Working Dilution (BF/DF) . |
---|---|---|---|---|---|
GAD65/67 | Glutamic acid decarboxylases 65 and 67 | Inhibitory, GABAergic neurons | Abcam (ab49832) | rb | n.a./100 |
GFAP | Glial fibrillary acidic protein | Astrocytes | Progen (GP52) | gp | 5.000/n.a. |
Iba1 | Ionized calcium-binding adaptor protein 1 | Microglia | Wako Chemicals (019-19741) | rb | 500/n.a. |
MAP2 | Microtubule-associated protein 2 | Neuronal cell bodies and dendrites | Abcam (ab183830) | rb | n.a./500 |
NF200 | Neurofilament heavy chain 200kD | Neuronal cell bodies and axons | Abcam (ab8135) | rb | n.a./100 |
PGP9.5 | Protein-gene-product 9.5 | Excitatory neurons | Cedarlane (CL95101) | rb | n.a./1.000 |
SOD1 | Superoxide dismutase 1 | Human SOD1 | Santa Cruz Biotech. (sc-8637) | gt | 1.000/500 |
gp: guinea pig, gt, goat; ms, mouse; n.a., not applicable; rb: rabbit. Company details: Abcam, Cambridge, UK; Cedarlane, Burlington, Ontario, Canada; Progen Biotechnik GmbH, Heidelberg, Germany; Santa Cruz Biotech. Inc., Dallas, TX; Wako Chemicals, Neuss, Germany.
Antibody (Abbr.) . | Target PROTEIN . | Specificity . | Source . | Donor Species . | Working Dilution (BF/DF) . |
---|---|---|---|---|---|
GAD65/67 | Glutamic acid decarboxylases 65 and 67 | Inhibitory, GABAergic neurons | Abcam (ab49832) | rb | n.a./100 |
GFAP | Glial fibrillary acidic protein | Astrocytes | Progen (GP52) | gp | 5.000/n.a. |
Iba1 | Ionized calcium-binding adaptor protein 1 | Microglia | Wako Chemicals (019-19741) | rb | 500/n.a. |
MAP2 | Microtubule-associated protein 2 | Neuronal cell bodies and dendrites | Abcam (ab183830) | rb | n.a./500 |
NF200 | Neurofilament heavy chain 200kD | Neuronal cell bodies and axons | Abcam (ab8135) | rb | n.a./100 |
PGP9.5 | Protein-gene-product 9.5 | Excitatory neurons | Cedarlane (CL95101) | rb | n.a./1.000 |
SOD1 | Superoxide dismutase 1 | Human SOD1 | Santa Cruz Biotech. (sc-8637) | gt | 1.000/500 |
Antibody (Abbr.) . | Target PROTEIN . | Specificity . | Source . | Donor Species . | Working Dilution (BF/DF) . |
---|---|---|---|---|---|
GAD65/67 | Glutamic acid decarboxylases 65 and 67 | Inhibitory, GABAergic neurons | Abcam (ab49832) | rb | n.a./100 |
GFAP | Glial fibrillary acidic protein | Astrocytes | Progen (GP52) | gp | 5.000/n.a. |
Iba1 | Ionized calcium-binding adaptor protein 1 | Microglia | Wako Chemicals (019-19741) | rb | 500/n.a. |
MAP2 | Microtubule-associated protein 2 | Neuronal cell bodies and dendrites | Abcam (ab183830) | rb | n.a./500 |
NF200 | Neurofilament heavy chain 200kD | Neuronal cell bodies and axons | Abcam (ab8135) | rb | n.a./100 |
PGP9.5 | Protein-gene-product 9.5 | Excitatory neurons | Cedarlane (CL95101) | rb | n.a./1.000 |
SOD1 | Superoxide dismutase 1 | Human SOD1 | Santa Cruz Biotech. (sc-8637) | gt | 1.000/500 |
gp: guinea pig, gt, goat; ms, mouse; n.a., not applicable; rb: rabbit. Company details: Abcam, Cambridge, UK; Cedarlane, Burlington, Ontario, Canada; Progen Biotechnik GmbH, Heidelberg, Germany; Santa Cruz Biotech. Inc., Dallas, TX; Wako Chemicals, Neuss, Germany.
Double Immunofluorescence
To determine the cell type affected by SOD1G93A-related vacuolization standard double immunofluorescence against several neuronal marker proteins was performed on the paraffin sections as follows. After deparaffination and blocking procedures (see above), goat anti-hSOD1 and a second primary antibodies directed against a marker protein and derived in a different donor species (Table) were coapplied in PBS/1% BSA and incubated overnight at 4 °C, followed by 2 hours at 37 °C. After extensive washing in distilled water followed by PBS, hSOD1 immunoreactions were visualized with antigoat secondary antibodies labeled with Alexa Fluor 647 (MoBiTec, Göttingen, Germany), diluted 1:200 in 1% BSA/PBS. The other antibody was visualized by a two-step procedure using a species-specific biotinylated secondary antibody (Dianova), diluted 1:200 in 1% BSA/PBS followed by streptavidin conjugated with Alexa Fluor 488, diluted 1:100 in 1% BSA/PBS. Incubation times were 45 minutes with the biotinylated secondary antibody only, followed by 2-hour incubation with a mixture of fluorochrome-conjugated secondary antibody and streptavidin. Immunofluorescence signals were documented as digitized false color images (8-bit tiff format) with an Olympus BX50WI confocal laser scanning microscope (Olympus Optical, Hamburg, Germany) and Olympus Fluoview 2.1 software.
Quantitative Assessment of Neuroinflammation
Astrogliosis was evaluated in 3–4 SOD1 end-stage mice and in 4 age-matched WT mice. Six tissue sections, separated at least by 35 µm, taken from each mouse were processed with glial fibrillary acidic protein (GFAP) antibodies as described above. Under bright-field illumination and ×200 magnification, immunoreactions in the ganglion cell layer (rGCL) of the retina and in the external plexiform layer (EPL) of the olfactory bulb were documented as black/white images in TIF format. At least 4 nonoverlapping images were taken from each tissue section. Using ImageJ software (NIH, Bethesda, MD), the area [%] covered by GFAP immunoreactivity was determined by applying a constant threshold.
Microgliosis was evaluated on the same number of tissue sections as described above, using ionized calcium-binding adaptor protein 1 (Iba1) antibodies. The numbers of microglial cell bodies were determined in the retina (i.e. in rGCL, EPL, inner nuclear layer [INL], and internal plexiform layer [IPL]), and in the olfactory bulb (in the EPL) by microscopic visual inspection at ×400 magnification. Subsequently, these cell counts were put in relation to the measured retina length, or olfactory bulb EPL area, respectively.
Means, standard deviations, and standard errors of the means (SEM) were calculated from the data of each mouse, and subjected to statistical analysis with unpaired t-test using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA). A significance level of p ≤ 0.05 was applied.
RESULTS
Early and Spatially Restricted Neuropathology in the Olfactory Bulb of SOD1G93A Mice
The mouse olfactory bulb is organized into discrete layers (Fig. 1A). In the outermost glomerular layer (GL), axons from the receptor neurons in the olfactory epithelium synapse on dendrites of mitral and tufted cells. While tufted cells are located within the EPL, mitral cells form a separate cell layer. The axons of both mitral and tufted cells cross the internal plexiform layer (IPL) and the granule cell layer (oGCL), which contains a variety of inhibitory neurons, and leave the olfactory bulb to higher olfaction areas via the olfactory tract.
When compared to WT (Fig. 1A), Giemsa staining revealed severe vacuolization pathology in the intermediate and deep EPL of end-stage SOD1G93A mice (Fig. 1B). To investigate if this vacuolization reflected an ALS-related pathology we performed an immunohistochemical time course analysis using hSOD1 as a marker. While generally absent from WT mice (shown here for P60, Fig. 1C), hSOD1 immunoreactivity first displayed as small dots in the presymptomatic stage at P60 in the EPL of SOD1G93A mice (Fig. 1D). These hSOD1-immunoreactive structures then increased in size during disease progression, shown here for disease onset at P90 (Fig. 1E), with some of them developing a visible lumen and reaching a diameter of up to 10 µm at disease end-stage (Fig. 1F, G). In addition to the severe vacuolization in the intermediate and deep EPL, hSOD1 immunostaining also visualized a few vacuoles in the GL and oGCL (arrows in Fig. 1E, F). In contrast to the massive, yet spatially restricted pathology in the olfactory bulb, obvious vacuolization was absent from subsequent aspects of the olfactory pathway up until diseases end-stage (Fig. 1H), including the lateral olfactory tract (Fig. 1I), the anterior olfactory nucleus (Fig. 1J), the piriform cortex (Fig. 1K), and the olfactory tubercle (Fig. 1L). Of note, the abundance, size, and distribution of the vacuoles was similar in all animals investigated for a respective time point.
Early and Spatially Restricted Neuropathology in the Retina of SOD1 Mice
Like the olfactory bulb, the mouse retina is structured into distinct layers (Fig. 2A). Light is transduced into electrical signals by rod and cone photoreceptor cells, whose cell bodies build the outer nuclear layer. Within the outer plexiform layer they transmit their signal onto bipolar cells, whose cell bodies build the INL. Bipolar cell axons reach into the inner plexiform layer (IPL), where the signal is relayed onto retinal ganglion cells. The retinal ganglion cells form a separate cell layer (i.e. the rGCL) and their axons converge to the optic nerve that relays in the dorsal lateral geniculate nucleus of the thalamus. From there, the fourth neuron of the visual pathway projects into the primary visual cortex.
There were no obvious differences visible on the histological level when comparing retinae from WT (Fig. 2A) and end-stage SOD1G93A (Fig. 2B) mice in Giemsa-stained sections. However, a time course analysis of hSOD1 immunohistochemistry (Fig. 2C–F) revealed the appearance of immunoreactive structures, particularly in the IPL, of SOD1G93A mice retinae. As for the olfactory bulb, these vacuoles were absent in WT (Fig. 2C), however, very rare and tiny in presymptomatic SOD1G93A mice at P50 (not shown), and became more frequent in SOD1G93A mice at P70 (Fig. 2D). The vacuole sizes constantly increased over time, shown here for P90 (Fig. 2E), and finally showed a lumen and reached a diameter of ≤10 µm at disease end-stage (Fig. 2F). In addition to the severe vacuolization in the IPL, immunostaining against hSOD1 also visualized a few vacuoles in the rGCL and in the INL (arrows in Fig. 2F). Furthermore, a moderate number of large vacuoles with a diameter up to 10 µm were found in the optic nerve papilla (Fig. 2G). However, these vacuoles located mostly at the rim of the optic nerve papilla and thus seemed to be an extension of the INL, while vacuoles were rarely found in the midline where the axons of the retinal ganglion cells converge and merge into the optic nerve (Fig. 2G). Similarly, no vacuolization was present in the optical tract (Fig. 2H) and in higher order areas of the visual pathway (identified here in a Giemsa-stained section, Fig. 2I), i.e. the dorsal lateral geniculate nucleus of the thalamus (Fig. 2J) and the primary visual cortex (Fig. 2K). As in the olfactory bulb, time course, distribution, and morphology of the vacuoles were nearly identical in all animals investigated.
Dendrites of Excitatory Neurons Are the Source of SOD1G93A-Related Pathology in the Olfactory Bulb and Retina
We next aimed to identify the neuron type showing vacuolization in the EPL of the olfactory bulb and the IPL of the retina, respectively. Besides the cell bodies and neurites of tufted cells and interneurons, the EPL of the olfactory bulb harbors the dendritic trees of mitral cells (24). Also dendrites of inhibitory granule cells originating in the oGCL enter the EPL. To elucidate in which type of neurite the vacuoles are located in, we performed a double immunofluorescence analysis of hSOD1 with the dendrite marker, microtubule associated protein 2 (MAP2), and the axonal marker, neurofilament 200 (NF200), at disease end-stage (Fig. 3). All hSOD1-immunoreactive vacuoles colocalized with MAP2 (Fig. 3A), while no spatial association between hSOD1 and NF200 immunoreactivity was found (Fig. 3B).
In the retina, the IPL consists of the dendrites of the retinal ganglion cells as well as of the axonal processes of the bipolar cells located in the INL. In addition, the INL harbors a high number of amacrine cells, which by definition lack the axon, but send highly arborized dendritic trees into the IPL. The majority of amacrine cells are inhibitory, making contacts with both bipolar and ganglion cells. As for the olfactory bulb’s EPL, all hSOD1-immunoreactive vacuoles in the retina’s IPL costained for MAP2 (Fig. 3C), but not NF200 (Fig. 3D).
To determine if the dendritic vacuoles belonged to excitatory or inhibitory neurons, protein gene product 9.5 (PGP9.5, a marker for excitatory neurons and thus tufted and mitral cells in the olfactory bulb [25] and retinal bipolar and ganglion cells), and glutamic acid decarboxylase (GAD65/67, a marker for inhibitory neurons and thus olfactory granule cells and retinal amacrine cells) were combined with the detection of hSOD1. In the olfactory bulb, PGP9.5 colocalized with the rim of the vacuoles (Fig. 4A–C), while GAD65/67-positive structures appeared to be in close proximity with the vacuoles but not colocalizing with them (Fig. 4D–F). In addition, we stained with markers for some olfactory bulb inhibitory interneuron subtypes that either reside within the EPL, or project into this layer. These included tyrosine hydroxylase, choline acetyltransferase, vasoactive intestinal peptide, somatostatin, calcitonin gene-related peptide, and corticotropin-releasing factor. This analysis did not reveal a staining pattern comparable to that of SOD1 (data not shown).
Similarly, double-immunofluorescence of hSOD1 against PGP9.5 in the retina revealed partial colocalization with the vacuole rims (Fig. 4G–I), while GAD65/67 appeared to be in direct proximity with the hSOD1-immunoreactive vacuoles but without any colocalization (Fig. 4J–L). Again, staining for inhibitory amacrine cell subtypes with tyrosine hydroxylase, choline acetyltransferase, chromogranin A, pituitary adenylate cyclase-activating peptide, substance P, and vasoactive intestinal peptide, was found unrelated to the SOD1-related vacuolization pathology (data not shown).
Vacuolization in the Olfactory Bulb and Retina Is Not Accompanied by a Neuroinflammatory Reaction
In many brain areas of SOD1G93A mice, and especially in vulnerable motor nuclei and spinal cord, vacuolization and hence neurodegeneration is accompanied by a neuroinflammatory reaction (22, 26). On the cellular level, neuroinflammation displays as morphology changes, i.e. hypertrophy of cell bodies, together with shortening and thickening of cell processes. To determine if a neuroinflammatory reaction also paralleled vacuolization in the EPL of the olfactory bulb and in the IPL of the retina, we performed immunohistochemistry for GFAP, an astrocyte marker, and for Iba1, a microglia marker.
In WT mice (Fig. 5A), sparse GFAP immunoreactivity was detected in the EPL of the olfactory bulb. Despite massive SOD1-related vacuolization (Fig. 5B), there was no indication for astrocyte hypertrophy in SOD1G93A end-stage mice, as determined by the area covered with GFAP immunoreactivity (WT = 3.29% ± 2.06%; SOD1 = 2.92% ± 2.28%; p = n.s.; Fig. 5C). A similar observation was made in the retina, where astrocytes reside in the rGCL (27, 28). Both in WT (Fig. 5D) and in SOD1G93A mice at disease end-stage (Fig. 5E), astrocyte morphologies were found to be similar, and GFAP staining was restricted to the rGCL. There was no indication for astrocyte hypertrophy (WT = 5.03% ± 0.88%; SOD1 = 7.12% ± 0.21%; p = n.s.; Fig. 5F), for an induction of GFAP expression in Müller cells (29), or for astrocyte penetration into deep retinal layers (30).
Iba1-immunoreactive microglia displayed as small cells with fine, ramified processes in the EPL of the olfactory bulb, both in WT (Fig. 5G), and in SOD1G93A mice at disease end-stage (Fig. 5H). There was no indication for an increase in microglia cell numbers in SOD1G93A mice compared to WT (WT = 65.58 ± 8.16 cells/mm2; SOD1 = 51.63 ± 4.87 cells/mm2; p = n.s.; Fig. 5I). Microglia in the retina are sparse and predominantly settle in the plexiform layers (31). Again, microglia from WT (Fig. 5J) and from SOD1G93A mice (Fig. 5K) was found indifferent in morphology and number (WT = 3.27 ± 0.95 cells/mm; SOD1 = 3.14 ± 1.23 cells/mm; p = n.s.; Fig. 5L).
DISCUSSION
Neuropathology in the Olfactory System
In the olfactory bulb of SOD1G93A mice, the vast majority of hSOD1-immunoreactive vacuoles localized to the lower half of the EPL, and only few vacuoles were detectable in the GL and the oGCL. Subsequent nerve fiber tracts and olfaction relay stations inside and outside the olfactory bulb were not overtly affected by vacuolization pathology. Notably, vacuolization initiated at a presymptomatic stage. A close spatial relation of the hSOD1-immunoreactive vacuoles with MAP2 and with PGP9.5 was indicative of a presence in dendrites of excitatory neurons, while this was not the case with NF200, GAD65/67, and several inhibitory interneuron markers. Mitral cells extend a primary dendrite into the GL, and many secondary dendrites into the deep and intermediate EPL. The sparse vacuolization pathology in the GL suggests that mitral primary dendrites are not affected in general, but points to a more restricted pathology in secondary dendrites. Alternatively, pathology may be restricted to the proximal compartments of both dendrite types and does not spread to more distal parts. Since the majority of tufted cells extend their secondary dendrites into the superficial/outer EPL, one can conclude that this cell type is not affected, or only to a minor extent. Nevertheless, mitral cell secondary dendrites make dendrodendritic interactions with granule cells and other interneurons, a process called lateral inhibition (32) that might explain the close proximity between the vacuoles (located in mitral cell dendrites) and GAD65/67-immunoreactive structures (i.e. the dendrites of inhibitory granule cells and interneurons). Future experiments need to address if this pathology has functional consequences, e.g. in odor discrimination related to food consumption and/or social interaction.
Unfortunately, information about olfactory histopathology in human sporadic and familial forms of ALS is limited. Hawkes et al (12) found that 95% of olfactory neurons (i.e. from anterior olfactory nucleus and other principal neurons of the olfactory bulb and tract) contained lipofuscin inclusions in all the 58 patients investigated, compared to only 50% of the neurons of controls. In a more recent study (7), a high abundance of TDP-43-positive inclusions was detected in higher order olfactory regions of the limbic system, but also as some perinuclear inclusions in granule cells of the olfactory bulb in about 50% of the investigated ALS patients, which was suggestive of a posterior to anterior spread of pathology in the olfactory system. Besides postmortem analysis of patient tissues, odor tests revealed that ALS patients may be slightly, but sometimes also severely compromised in odor perception depending on the respective disease subtype (7, 12–15). Thus, we propose that olfaction tests should be performed routinely in patients, and subsequently compared with the respective level of postmortem brain pathology. Such an investigation may reveal important correlations between ALS subtypes and the manifestation of sensory, here olfactory neuropathology (33). This information could be used to determine the underlying disease causes by comparing differences in molecular, physiological or anatomical features of ALS vulnerable and resistant neuron types within the respective ALS subtypes with or without olfactory impairment.
Neuropathology in the Visual System
In the retinae of the investigated SOD1G93A mice, hSOD1-immunoreactive vacuoles were found largely restricted to the lower two thirds of the IPL, and only rarely seen in INL or rGCL. Again, vacuoles appeared at presymptomatic stages, and double-immunofluorescence analysis revealed a localization of these vacuoles to dendritic processes of excitatory neurons. Downstream fiber tracts (the optic nerve and optic tract) and relay stations of the visual system (DLG and V1) were unaffected. This suggests that vacuolization pathology is restricted to dendrites of retinal ganglion cells. Recently, a comparable pathology has been described in a transgenic ALS model that expresses a dysfunctional ubiquilin2 gene, the UBQLN2P497H mouse (34). Here, some ubiquilin2-immunoreactive deposits were present in the IPL, while they were rare in the outer plexiform layer. In addition, p62- and ubiquitin-positive inclusions were reported to be present in the INL, IPL and rGCL layers in the retina of a patient harvesting a C9orf72 mutation that was associated with an impairment of contrast sensitivity (11). Using double-immunofluorescence, the authors suggested a specific type of bipolar cell being the affected neuron in the INL, which is in contrast to our findings in SOD1G93A mice. Thus, retinal pathology seems not restricted to the SOD1-based familial forms of ALS, and highlights the necessity to also investigate retinae from transgenic mice expressing other disease-causing mutation, as well as from human patient postmortem tissue to obtain a complete picture of retinal involvement in ALS.
Along this line, using optical coherence tomography, a thinning of select retinal layers has been discerned in ALS patients in vivo. With reductions in the thickness of the retinal nerve fiber layer being the most consistent finding of these studies (8–10), additional thinning of the INL was found in a subset (9, 10), while such pathology was completely absent in another study (35). Unfortunately, with this technique the IPL can only be assessed together with the rGCL, which might mask additional effects on ganglion cell dendrites and/or soma. However, these studies also suggest the ganglion cell as the most vulnerable neuron type in the retina in context of ALS pathology. Furthermore, the differences in the results obtained from different patient cohorts (in which some familial cases were included while excluded in others) highlight the necessity to take different ALS subtypes into account. For example, olfactory impairment reached only significance for patients with bulbar, but not spinal onset (12), while shrinkage of the INL was only significant for spinal, but not bulbar onset patients (9). Accordingly, a C9orf72 mutation and other causes with comparable mechanisms might lead to degeneration of bipolar neurons and thus thinning of the INL, while SOD1 mutations and other comparable causes predominantly affect the ganglion cells and thus result in pathology of the IPL and/or the retinal nerve fiber layer. Nonetheless, possible visual perturbations in ALS patients should be monitored on a regular basis, and may serve as a prognostic marker for disease progression in the presence or absence of a therapeutic intervention.
Dendropathy of Excitatory Neurons in Olfactory and Retinal Sensory Pathways
The restriction of vacuolization to the dendritic compartment of sensory neurons, described here for the first time, is an intriguing finding, and is in contrast to the pathology found for somatomotor neurons in both brainstem and spinal cord. There, vacuoles appear throughout the degenerating neuron, i.e. in dendrites, axons, and in the soma (21, 36, 37). In case of the latter, one could observe the accumulation of vacuoles that leads to a disintegration of cell boundaries and thus degeneration of the neuron between P60 and end-stage, when the vacuoles reached diameters of up to 20 µm (36, 37). The maximum diameter of the vacuoles was approximately 10 µm for both olfactory bulb (Fig. 1F) and retina (Fig. 2F), and the soma of mitral and retinal ganglion cells appeared healthy even in end-stage SOD1G93A mice (Figs. 1F and 2F), suggesting that these neurons do not degenerate and die, at least during the time frame SOD1G93A mice survive.
While vacuole numbers and sizes vary greatly between areas affected by this particular neuropathology in SOD1G93A mice (17, 22), the beginning of pathological changes in the mitochondrial compartment occurs simultaneously around P60 throughout the CNS in spinal (36) and bulbar (37) somatomotor neurons, extrapyramidal motor areas such as the substantia nigra or red nucleus (22), and is a common characteristic of human ALS (38, 39). This implies that all these functionally and morphologically different neuron types are primarily affected by the not yet fully elucidated disease-causing mechanisms induced by the SOD1 mutation (or the hSOD1 overexpression, which cannot be fully excluded as molecular cause in this mouse model [19]). However, while in somatomotor neurons this pathomechanism results in degeneration of the whole neuron and thereby loss of function, the same molecular challenges in retinal and olfactory neurons described here may only lead to a dendropathy that leaves the neuron viable and presumably also functional. A similar scenario is found in the Opa1+/− mouse model of autosomal dominant optic atrophy, where reductions in OPA1 protein abundance perturb mitochondrial dynamics and result in dendritic pruning of retinal ganglion cells (40).
Thus, the dendritic pathology seen in SOD1G93A mice may precede the onset of clinically relevant visual and olfactory impairments in the absence of significant cell loss. Furthermore, this dendropathy occurred unrelated to a local neuroinflammatory reaction, which is in contrast to somatomotor regions where the secretion of mutant SOD1 proteins by neurons is thought to induce a fatal switch from neuroprotective to neurotoxic neuroinflammation (41, 42). Absence of morphological changes of astrocytes and microglia shown here however does not exclude functional changes, such as inflammation-related changes in expression levels of cytokines. However, as stated above, vacuolization in other brain regions begins at the same time point (around P50), but is followed by changes in microglia morphology already at P60 (21, 22, 36). The fact that these results are obtained from similar studies, and in the end-stage olfactory bulb in the very same animals (22), allows us to conclude that neuroinflammation in the two sensory areas analyzed is—if present at all—of a very alleviated or largely different (maybe even enduringly protective) phenotype.
Conclusions
The involvement of the anterior visual and olfactory pathways seems to be a more common feature in ALS than previously thought, with our study being the first to show that comparable pathologies are also present in the olfactory bulb and retina of the most frequently used ALS mouse model. The SOD1G93A model thus recapitulates the situation found in human ALS with great accordance, reassuring its feasibility and importance to elucidate the molecular mechanisms underlying this fatal disease (43). Furthermore, our study revealed a remarkable parallelism between neuropathology in the olfactory bulb and retina, i.e. affection of the dendritic compartment of excitatory projection neurons that, to our knowledge, has not yet been described for any other neurodegenerative condition.
Elaborating the molecular differences between these distinct neuron types with variable degrees of vulnerability and those neurons which do not show any vulnerability to the SOD1G93A mutation will lead to important insights into the mechanisms that cause neurodegeneration in ALS. Moreover, extending the investigations of the involvement of olfactory and retinal neurons to other ALS mouse models and to autopsy material of ALS patients will help to further elucidate phenotypic and ultimately mechanistic differences between the diverse ALS subtypes and to develop ALS subtype specific diagnostic and prognostic markers, and treatments.
ACKNOWLEDGEMENT
We thank Marion Zibuschka for excellent technical assistance.
REFERENCES
Author notes
Present address: Institute for Anatomy, University of Lübeck, Lübeck, Germany (CR).
Financial support: This work was supported by funds from the University Medical Center Giessen and Marburg (UKGM, Germany), by the P. E. Kempkes Foundation (University of Marburg, Germany), and by grants from the German Society for the Muscular Diseased (DGM, Freiburg, Germany).
Conflict of interest: The authors have no duality or conflicts of interest to declare.