Exciting Complexity: The Role of Motor Circuit Elements in ALS Pathophysiology

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the degeneration of both upper and lower motor neurons. Despite decades of research, we still to date lack a cure or disease modifying treatment, emphasizing the need for a much-improved insight into disease mechanisms and cell type vulnerability. Altered neuronal excitability is a common phenomenon reported in ALS patients, as well as in animal models of the disease, but the cellular and circuit processes involved, as well as the causal relevance of those observations to molecular alterations and final cell death, remain poorly understood. Here, we review evidence from clinical studies, cell type-specific electrophysiology, genetic manipulations and molecular characterizations in animal models and culture experiments, which argue for a causal involvement of complex alterations of structure, function and connectivity of different neuronal subtypes within the cortical and spinal cord motor circuitries. We also summarize the current knowledge regarding the detrimental role of astrocytes and reassess the frequently proposed hypothesis of glutamate-mediated excitotoxicity with respect to changes in neuronal excitability. Together, these findings suggest multifaceted cell type-, brain area- and disease stage- specific disturbances of the excitation/inhibition balance as a cardinal aspect of ALS pathophysiology.

Keywords: Amyotrophic lateral sclerosis; astrocytes; excitability; excitotoxicity; interneurons; lower motor neurons; neural circuits; upper motor neurons.

FIGURE 1

M1 circuitry and pathophysiological changes in ALS. Local (glutamatergic, excitatory) input to upper motor neurons (UMN, brown) is mainly provided by upstream LII/III pyramidal neurons (PN, light brown) and modulated by astrocytes (AS, gray). Parvalbumin (PV, magenta), somatostatin (SST, blue) and vasoactive intestinal peptide (VIP, green) interneurons provide GABAergic input within M1. PV and SST target PN, including UMNs, as well as inhibit each other (inhibition of PV by SST more frequent). VIP are disinhibitory by synapsing on PV and SST. Long-range input to M1 originates from cortical and subcortical structures: thalamus (TH), primary motor cortex (M1), secondary motor cortex (M2), somatosensory cortex (SS), auditory cortex (AUD). Neuromodulatory input stems from: the locus coeruleus [LC, releasing norepinephrine (NE)], ventral tegmental area [VTA, releasing dopamine (DA)], dorsal raphe [DR, releasing serotonin (5-HT)], tuberomammillary nucleus [TMN, releasing histamine (HA)] and basal forebrain [BF, acetylcholine (ACh)]. UMN project to the spinal cord, brainstem and send axon collaterals to the thalamus and striatum. Pathological changes (light brown filled circles) are identified throughout the M1 microcircuitry: Structural changes, e.g., altered spine density and dendritic regression are observed on apical dendrites of LII/III PN^1,2 and on apical and basal dendrites of UMN^1,2, along with a reduction in overall number⁷ and soma size^7,8,12. UMN are hyperexcitable^3,4,5,6, but don’t display overall activity changes⁵. Interneuron density was affected: while PV and SST are reduced in Wobbler mice⁹, density of all three interneuron subtypes remained unchanged in SOD1^G93A mice^8,10. Excitability of PV and SST was altered differentially. While hyperexcitability was observed in PV of SOD1^G93A mice⁵, hypoexcitable PV were accompanied by hyperexcitable SST in TDP-43^A315T mice¹¹. ¹(Fogarty et al., 2016b); ²(Fogarty et al., 2015); ³(Fogarty et al., 2016a); ⁴(Pieri et al., 2009); ⁵(Kim et al., 2017); ⁶(Saba et al., 2016); ⁷(Zang and Cheema, 2002); ⁸(Özdinler et al., 2011); ⁹(Nieto-Gonzalez et al., 2011); ¹⁰(Clark et al., 2017); ¹¹(Zhang et al., 2016); ¹²(Gautam et al., 2016).

FIGURE 2

ALS-associated alterations in ventral spinal cord circuitry. LMN receive inhibitory input via Ia-IN, Ib-IN, and RC, and excitatory inputs from cortiospinal tract (UMN), II-IN and SN. γ-motor neurons, which are spared in ALS, do not receive direct inputs from Ia-SN. Excitatory inputs to LMNs via Ia afferent terminals are controlled by PI-IN. Both excitatory and inhibitory inputs are tightly regulated by the proprioceptive feedback provided by sensory afferents (Ia, Ib and II-SN) and astrocytes. Axonal hyperexcitability and hypoexcitability are reported in ALS patients. Decreased RC synapses on LMN and lower number of RC is reported. LMN hypoexcitability is present in vivo in SOD1^G93A tg mouse model. Ia-SN neurons exhibit irregular firing patterns as an indication of their altered excitability/activity. Cholinergic C-bouton number is decreased in sALS patients, but C-boutons are enlarged especially onto vulnerable FF LMN in SOD1^G93A tg mice. Protein and mRNA levels of ChAT are decreased in spinal cord of ALS patients. Reduced ChAT expression is reported in II-IN and C-boutons on MN of SOD1^G93A tg mice. Serotonergic boutons on LMN are increased in low-copy SOD1^G93A tg mice, whereas serotonergic neurons in brainstem degenerate in both ALS patients and SOD1^G86R tg (not shown). Please note that monosynaptic connections between UMN and LMN are only present in humans. Neuromodulatory synapses are depicted as one (somata located in brainstem). CPGs and descending reticulospinal tract projections to LMN via commissural INs are not depicted for simplicity. Studies with unspecified type of ALS are referred to as (ALS). AP, action potential; AS, astrocytes; CMAP, compound muscle action potential; ChAT, choline acetyltransferase; CPG, central pattern generator; DD, double-discharge; fALS, familial ALS; FF, fast-fatigable; FP, fasciculation potential; FR, fast-resistant; gamma (γ)-motor neuron, γ-MN; GAD65/67, glutamic acid decarboxylase 65/67; Glu, glutamate; GlyT2, glycine transporter 2; Ia-/Ib-IN, class Ia/Ib spinal interneuron; II-IN, class II spinal interneuron; LMN, lower motor neuron; MU, motor unit; NE, norepinephrine; Ia-/Ib-SN, class Ia/Ib sensory neuron; II-SN, class II sensory neuron; PI-IN, presynaptic inhibitory interneuron; RC, Renshaw cell; sALS, sporadic ALS; SOD1, superoxide dismutase 1; S, slow; TEd, depolarizing threshold electrotonus; TEh, hyperpolarizing threshold electrotonus; τ_SD, strength-duration constant, UMN, upper motor neuron; VGAT, vesicular GABA transporter; VGLUT, vesicular glutamate transporter 2; 5-HT, serotonin. ¹(Kostera-Pruszczyk et al., 2002); ²(Kanai et al., 2006); ³(Vucic and Kiernan, 2006); ⁴(Vucic et al., 2009); ⁵(Bostock et al., 1995); ⁶(Horn et al., 1996); ⁷(Mogyoros et al., 1998); ⁸(Tamura et al., 2006); ⁹(Nakata et al., 2006); ¹⁰(Piotrkiewicz et al., 2008); ¹¹(de Carvalho and Swash, 2013); ¹²(Raynor and Shefner, 1994); ¹³(Hayashi et al., 1981); ¹⁴(Whitehouse et al., 1983); ¹⁵(Howells et al., 2018); ¹⁶(Delestrée et al., 2014); ¹⁷(Martínez-Silva et al., 2018); ¹⁸(Chang and Martin, 2009); ¹⁹(Chang and Martin, 2011a); ²⁰(Chang and Martin, 2011b); ²¹(Seki et al., 2019); ²²(Heads et al., 1991); ²³(Hammad et al., 2007); ²⁴(Pugdahl et al., 2007); ²⁵(Sangari et al., 2016); ²⁶(Guo et al., 2009); ²⁷(Sassone et al., 2016); ²⁸(Nagao et al., 1998); ²⁹(Oda et al., 1995); ³⁰(Virgo et al., 1992); ³¹(Casas et al., 2013); ³²(Pullen and Athanasiou, 2009); ³³(Saxena et al., 2013); ³⁴(Dentel et al., 2012); ³⁵(Hossaini et al., 2011); ³⁶(Sunico et al., 2011).

FIGURE 3

ALS-associated changes in LMN excitability assessed in vitro. Findings obtained through recordings of LMN (brown) in embryonic/neonatal cell culture, IPSC-derived LMN culture or spinal cord slice culture are shown. Data indicative of hyperexcitability is shown in red, hypoexcitability in blue and no change in green. Abbreviations: C9orf72, chromosome 9 open reading frame 72; DR, delayed-rectifying; FI, fast -inactivating; F-I, frequency-current; FUS, fused in sarcoma; IPSC, induced pluripotent stem cell; LMN, lower motor neuron; RMP, resting membrane potential; SOD1, superoxide dismutase 1; TDP-43, TAR DNA-binding protein 43; VG, voltage-gated. ¹(Wainger et al., 2014); ²(Devlin et al., 2015); ³(Sareen et al., 2013); ⁴(Naujock et al., 2016); ⁵(Pieri, 2003); ⁶(Kuo et al., 2004); ⁷(Kuo et al., 2005); ⁸(Bories et al., 2007); ⁹(van Zundert et al., 2008); ¹⁰(Quinlan et al., 2011); ¹¹(Martin et al., 2013); ¹²(Leroy et al., 2014); ¹³(Pambo-Pambo et al., 2009); ¹⁴(Jiang et al., 2017); ¹⁵(Chang and Martin, 2011a); ¹⁶(Delestrée et al., 2014).

FIGURE 4

Altered neuron-astrocyte interaction in ALS. Astrocytes lose homeostatic function through multiple proposed molecular dysregulations. Astrocytic EAAT2 downregulation results in increased synaptic glutamate (red dots) and enhanced postsynaptic NMDAR and AMPAR activation. Moreover, astrocyte-mediated K⁺ clearance is disrupted due to downregulation of Kir4.1 and upregulation of co-localized AQP4 channels. Astrocyte-neuron lactate shuttle is impaired as a result of reduced MCT4 expression leading to lower extracellular lactate concentration (Ferraiuolo et al., 2011). Astrocytes gain toxic properties by generating factors such as ROS, NOX2, and iNOS (Marchetto et al., 2008), NOS or TGF-β1 (Endo and Yamanaka, 2015). In addition, they secrete proinflammatory cytokines IFN-γ and IFN-α/β (Aebischer et al., 2011) and upregulate PGD2 receptor expression. Glutamate-excitotoxicity is aggravated by increased levels of D-Serine (pink dots) due to reduced degradation caused by DAO downregulation in astrocytes. Furthermore, astrocytes also display Ca²⁺ dyshomeostasis in the cytosol and ER. Connectivity between astrocytes is altered by elevated expression of the gap junction protein Cx43. Blue arrows indicate downregulation, whereas red arrows show upregulation. AC, adenylyl cyclase; ADP, adenosine diphosphate; AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; AQP4, aquaporin-4; ASC1, astrocytic transporter SLC7A10; ATM, ataxia telangiectasia mutated; Ca²⁺, calcium ion; Cx43, connexin 43; EAAT2, excitatory amino acid transporter 2; glucose-6-P, glucose-6-phophate; GDNF, glial cell-derived neurotrophic factor; GPCRs, G-protein-coupled receptors; Gq, q subunit of heterotrimeric G protein; HCAR1, hydroxycarboxylic acid receptor 1; IFN-α, interferon alpha; IFN-β, interferon beta; IFN- γ, interferon gamma; iNOS, inducible nitric oxide synthase; K⁺, potassium ion; Kir4.1, inward-rectifying potassium channel 4.1; LDHB, lactate dehydrogenase B; MCT1, monocarboxylate transporter 1; MCT2, monocarboxylate transporter 2; MCT4, monocarboxylate transporter 4; MHC1, major histocompatibility complex class I; NADPH, nicotinamide adenine dinucleotide phosphate hydrogen; NCX, sodium-calcium exchanger; NH₄, ammonium; NMDAR, N-methyl-D-aspartate receptor; NO, nitric oxide; NOX2, NADPH oxidase 2; NOS, nitric oxide synthase; O₂, oxygen; ORAI: Ca²⁺ release-activated Ca²⁺ channel protein 1; P2XR, ionotropic receptors; PDG2, prostaglandin D₂ receptor; PMCA, plasma membrane Ca²⁺-ATPase; VGIC, voltage-gated ion channels.

FIGURE 5

Interaction and synergy of proposed cell-autonomous vs. non-cell-autonomous mechanisms contributing to MN degeneration. Schematic depicting interdependency of proposed mechanisms. Cell-autonomous mechanisms refer to changes that take place within the affected MN, e.g., altered receptor expression. Non-cell-autonomous mechanisms refer to changes occurring on cell types other than MN, causally involved in the degenerative process, e.g., increased excitation (purple), lack of inhibition (yellow) and dysfunctional glutamate-reuptake system involving astrocytes (green).