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Astrocytes make up 20-40% of glial cells within the central nervous system (CNS) and provide several crucial functions, ranging from metabolic and structural support to regulation of synaptogenesis and synaptic transmission. Although these cells are morphologically and functionally complex, astrocytes have been historically regarded as homogenous cell populations and studied as one population of cells. Fortunately, recent evidence in RNA profiling and imaging data has begun to refute this view. These studies suggest heterogeneity of astrocytes across brain regions, differing in many aspects such as morphology, function, physiological properties, developmental origins, and response to disease. Increased understanding of astrocyte heterogeneity is critical for investigations into the function of astrocytes in the brain and neuro-glia interactions. Furthermore, insights into astrocyte heterogeneity can help better understand their role in neurological disorders and potentially produce novel approaches to treating these diseases.

The most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic hexanucleotide repeat expansion in the C9orf72 gene. In disease, RNA transcripts containing this expanded region undergo repeat‐associated non‐AUG translation to produce dipeptide repeat proteins (DPRs), which are detected in brain and spinal cord of patients and are neurotoxic both in vitro and in vivo paradigms. We reveal here a novel pathogenic mechanism for the most abundantly detected DPR in ALS/FTD autopsy tissues, poly‐glycine‐alanine (GA). Previously, we showed motor dysfunction in a GA mouse model without loss of motor neurons. Here, we demonstrate that mobile GA aggregates are present within neurites, evoke a reduction in synaptic vesicle‐associated protein 2 (SV2), and alter Ca2+ influx and synaptic vesicle release. These phenotypes could be corrected by restoring SV2 levels. In GA mice, loss of SV2 was observed without reduction of motor neuron number. Notably, reduction in SV2 was seen in cortical and motor neurons derived from patient induced pluripotent stem cell lines, suggesting synaptic alterations also occur in patients., Poly‐glycine‐alanine (GA) aggregates present in neurons cause a reduction in synaptic release and loss of the synaptic vesicle protein SV2 prior to neuronal death. Restoring SV2 levels in these neurons reinstates synaptic release functions and rescues neuronal toxicity.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two apparently distinct neurodegenerative diseases, the former characterized by selective loss of motor neurons in the brain and spinal cord and the latter characterized by selective atrophy of frontal and temporal lobes. Over the years, however, growing evidence from clinical, pathological and genetic findings has suggested that ALS and FTD belong to the same clinic-pathological spectrum disorder. This concept has been further supported by the identification of the most common genetic cause for both diseases, an aberrantly expanded hexanucleotide repeat GGGGCC sequence located in a non-coding region of the gene C9orf72. Three hypotheses have been proposed to explain how this repeats expansion causes diseases: 1) C9orf72 haploinsufficiency-expanded repeats interfere with transcription or translation of the gene, leading to decreased expression of C9orf72 protein; 2) RNA gain of function-RNA foci formed by sense and antisense transcripts of expanded repeats interact and sequester essential RNA binding proteins, causing neurotoxicity; 3) Repeat associated non-ATG initiated (RAN) translation of GGGGCC repeat expansion-RAN translation of expanded sense and antisense repeats produces potential toxic dipeptide repeat protein (DPR). In this review, we assess current evidence supporting or arguing against each proposed mechanism in C9 ALS/FTD disease pathogenesis. Additionally, controversial findings are also discussed. Lastly, we discuss the possibility that the three pathogenic mechanisms are not mutually exclusive and all three might be involved in disease.

SummaryAberrant hexanucleotide repeat expansions in C9orf72 are the most common genetic change underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). RNA transcripts containing these expansions undergo repeat-associated non-ATG translation (RAN-T) to form five dipeptide repeat proteins (DPRs). DPRs are found as aggregates throughout the CNS of C9orf72-ALS/FTD patients, and some cause degeneration when expressed in vitro in neuronal cultures and in vivo in animal models. The spread of characteristic disease-related proteins drives the progression of pathology in many neurodegenerative diseases. While DPR toxic mechanisms continue to be investigated, the potential for DPRs to spread has yet to be determined. Using different experimental cell culture platforms, including spinal motor neurons derived from induced pluripotent stem cells from C9orf72-ALS patients, we found evidence for cell-to-cell spreading of DPRs via exosome-dependent and exosome-independent pathways, which may be relevant to disease.

Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

Disorders of the thyroid cause a multitude of neurological dysfunctions including depression, anxiety, and psychosis. Thyroid hormones have been primarily thought to act via genomic mechanisms throughout the organism; however, recently another mechanism has been proposed for the adult brain. We hypothesize that the thyroid hormone triiodothyronine (T3) acts directly on GABA(A) receptors via a mechanism similar to that of neurosteroids. Previous electrophysiology experiments on expressed recombinant GABA(A) receptors demonstrated an inhibition of GABA responses in the presence of T3, and, at much higher concentrations of T3 alone, a direct stimulation of receptor activity. Current experiments are being conducted to investigate stereoselective effects and interactions with other compounds known to bind directly to the GABA(A) receptor. using a GABA(A) receptor homology model based on the crystal structure of the related Glutamate-gated chloride channel, atomic resolution molecular dynamics simulations were conducted to analyze the possible interaction of T3 and the GABA(A) receptor. In simulations, T3 is stable in binding sites in the transmembrane domain of the GABA(A)receptor in a region that is associated with the activation by neurosteroids. Alchemical free energy perturbation calculations are underway to test the affinity of T3 for the binding site in the GABA(A) receptor. Our results provide strong evidence supporting earlier experimental findings indicating a role of T3 in regulating the activity of GABA(A) receptors in brain, while bringing additional insight into both the molecular binding mode and the mechanism of modulation.

SummaryExpanded GGGGCC (G4C2) nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granule formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms.