Breakdown of the central synapses in C9orf72-linked ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease that leads to the death of motor and cortical neurons. The clinical manifestations of ALS are heterogenous, and efficacious treatments to significantly slow the progression of the disease are lacking. Cortical hyper-excitability is observed pre-symptomatically across disease-causative genetic variants, as well as in the early stages of sporadic ALS, and typically precedes motor neuron involvement and overt neurodegeneration. The causes of cortical hyper-excitability are not yet fully understood but is mainly agreed to be an early event. The identification of the nucleotide repeat expansion (GGGGCC)n in the C9ORF72 gene has provided evidence that ALS and another neurodegenerative disease, frontotemporal dementia (FTD), are part of a disease spectrum with common genetic origins. ALS and FTD are diseases in which synaptic dysfunction is reported throughout disease onset and stages of progression. It has become apparent that ALS/FTD-causative genes, such as C9ORF72, may have roles in maintaining the normal physiology of the synapse, as mutations in these genes often manifest in synaptic dysfunction. Here we review the dysfunctions of the central nervous system synapses associated with the nucleotide repeat expansion in C9ORF72 observed in patients, organismal, and cellular models of ALS and FTD.

Targeting TNFα produced by astrocytes expressing amyotrophic lateral sclerosis-linked mutant fused in sarcoma prevents neurodegeneration and motor dysfunction in mice.

Genetic mutations that cause amyotrophic lateral sclerosis (ALS), a progressively lethal motor neuron disease, are commonly found in ubiquitously expressed genes. In addition to direct defects within motor neurons, growing evidence suggests that dysfunction of non-neuronal cells is also an important driver of disease. Previously, we demonstrated that mutations in DNA/RNA binding protein fused in sarcoma (FUS) induce neurotoxic phenotypes in astrocytes in vitro, via activation of the NF-κB pathway and release of pro-inflammatory cytokine TNFα. Here, we developed an intraspinal cord injection model to test whether astrocyte-specific expression of ALS-causative FUSR521G variant (mtFUS) causes neuronal damage in vivo. We show that restricted expression of mtFUS in astrocytes is sufficient to induce death of spinal motor neurons leading to motor deficits through upregulation of TNFα. We further demonstrate that TNFα is a key toxic molecule as expression of mtFUS in TNFα knockout animals does not induce pathogenic changes. Accordingly, in mtFUS-transduced animals, administration of TNFα neutralizing antibodies prevents neurodegeneration and motor dysfunction. Together, these studies strengthen evidence that astrocytes contribute to disease in ALS and establish, for the first time, that FUS-ALS astrocytes induce pathogenic changes to motor neurons in vivo. Our work identifies TNFα as the critical driver of mtFUS-astrocytic toxicity and demonstrates therapeutic success of targeting TNFα to attenuate motor neuron dysfunction and death. Ultimately, through defining and subsequently targeting this toxic mechanism, we provide a viable FUS-ALS specific therapeutic strategy, which may also be applicable to sporadic ALS where FUS activity and cellular localization are frequently perturbed.

A mouse model with widespread expression of the C9orf72-linked glycine-arginine dipeptide displays non-lethal ALS/FTD-like phenotypes.

Translation of the hexanucleotide G4C2 expansion associated with C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) produces five different dipeptide repeat protein (DPR) species that can confer toxicity. There is yet much to learn about the contribution of a single DPR to disease pathogenesis. We show here that a short repeat length is sufficient for the DPR poly-GR to confer neurotoxicity in vitro, a phenomenon previously unobserved. This toxicity is also reported in vivo in our novel knock-in mouse model characterized by widespread central nervous system (CNS) expression of the short-length poly-GR. We observe sex-specific chronic ALS/FTD-like phenotypes in these mice, including mild motor neuron loss, but no TDP-43 mis-localization, as well as motor and cognitive impairments. We suggest that this model can serve as the foundation for phenotypic exacerbation through second-hit forms of stress.

Synaptic dysfunction induced by glycine-alanine dipeptides in C9orf72-ALS/FTD is rescued by SV2 replenishment.

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.

White matter loss and oligodendrocyte dysfunction in HIV: A consequence of the infection, the antiretroviral therapy or both?

While the severe cognitive effects of HIV-associated dementia have been reduced by combined antiretroviral therapy (cART), nearly half of HIV-positive (HIV+) patients still suffer from some form of HIV-Associated Neurocognitive Disorders (HAND). While frank neuronal loss has been dramatically reduced in HAND patients, white matter loss, including dramatic thinning of the corpus callosum, and loss of volume and structural integrity of myelin persists despite viral control by cART. It remains unclear whether changes in white matter underlie the clinical manifestation seen in patients or whether they are the result of persistent viral reservoirs, remnant damage from the acute infection, the antiretroviral compounds used to treat HIV, secondary effects due to peripheral toxicities or other associated comorbid conditions. Both HIV infection itself and its treatment with antiretroviral drugs can induce metabolic syndrome, lipodystrophy, atherosclerosis and peripheral neuropathies by increased oxidative stress, induction of the unfolded protein response and dysregulation of lipid metabolism. These virally and/or cART-induced processes can also cause myelin loss in the CNS. This review aims to highlight existing data on the contribution of white matter damage to HAND and explore the mechanisms by which HIV infection and its treatment contribute to persistence of white matter changes in people living with HIV currently on cART.

Reduced sterol regulatory element-binding protein (SREBP) processing through site-1 protease (S1P) inhibition alters oligodendrocyte differentiation in vitro.

The formation of the myelin membrane of the oligodendrocyte in the CNS is a fundamental process requiring the coordinated synthesis of many different components. The myelin membrane is particularly rich in lipids, however, the regulation of this lipid synthesis is not understood. In other cell types, including Schwann cells, the myelin-forming cells of the PNS, lipid synthesis is tightly regulated by the sterol regulatory element-binding protein (SREBP) family of transcription factors, but this has not been previously shown in oligodendrocytes. We investigated SREBPs' role during oligodendrocyte differentiation in vitro. Both SREBP-1 and SREBP-2 were expressed in oligodendrocyte precursor cells and differentiating oligodendrocytes. Using the selective site-1 protease (S1P) inhibitor PF-429242, which inhibits the cleavage of SREBP precursor forms into mature forms, we found that preventing SREBP processing inhibited process growth and reduced the expression level of myelin basic protein, a major component of myelin. Further, process extension deficits could be rescued by the addition of exogenous cholesterol. Blocking SREBP processing reduced mRNA transcription and protein levels of SREBP target genes involved in both the fatty acid and the cholesterol synthetic pathways. Furthermore, de novo levels and total levels of cholesterol synthesis were greatly diminished when SREBP processing was inhibited. Together these results indicate that SREBPs are important regulators of oligodendrocyte maturation and that perturbation of their activity may affect myelin formation and integrity. Cover Image for this issue: doi: 10.1111/jnc.13781.

HIV Protease Inhibitors Alter Amyloid Precursor Protein Processing via ß-Site Amyloid Precursor Protein Cleaving Enzyme-1 Translational Up-Regulation.

Mounting evidence implicates antiretroviral (ARV) drugs as potential contributors to the persistence and evolution of clinical and pathological presentation of HIV-associated neurocognitive disorders in the post-ARV era. Based on their ability to induce endoplasmic reticulum (ER) stress in various cell types, we hypothesized that ARV-mediated ER stress in the central nervous system resulted in chronic dysregulation of the unfolded protein response and altered amyloid precursor protein (APP) processing. We used in vitro and in vivo models to show that HIV protease inhibitor (PI) class ARVs induced neuronal damage and ER stress, leading to PKR-like ER kinase-dependent phosphorylation of the eukaryotic translation initiation factor 2α and enhanced translation of ß-site APP cleaving enzyme-1 (BACE1). In addition, PIs induced ß-amyloid production, indicative of increased BACE1-mediated APP processing, in rodent neuroglial cultures and human APP-expressing Chinese hamster ovary cells. Inhibition of BACE1 activity protected against neuronal damage. Finally, ARVs administered to mice and SIV-infected macaques resulted in neuronal damage and BACE1 up-regulation in the central nervous system. These findings implicate a subset of PIs as potential mediators of neurodegeneration in HIV-associated neurocognitive disorders.

Cell-to-Cell Transmission of Dipeptide Repeat Proteins Linked to C9orf72-ALS/FTD.

Aberrant 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.

Altered Oligodendrocyte Maturation and Myelin Maintenance: The Role of Antiretrovirals in HIV-Associated Neurocognitive Disorders.

Despite effective viral suppression through combined antiretroviral therapy (cART), approximately half of HIV-positive individuals have HIV-associated neurocognitive disorders (HAND). Studies of antiretroviral-treated patients have revealed persistent white matter abnormalities including diffuse myelin pallor, diminished white matter tracts, and decreased myelin protein mRNAs. Loss of myelin can contribute to neurocognitive dysfunction because the myelin membrane generated by oligodendrocytes is essential for rapid signal transduction and axonal maintenance. We hypothesized that myelin changes in HAND are partly due to effects of antiretroviral drugs on oligodendrocyte survival and/or maturation. We showed that primary mouse oligodendrocyte precursor cell cultures treated with therapeutic concentrations of HIV protease inhibitors ritonavir or lopinavir displayed dose-dependent decreases in oligodendrocyte maturation; however, this effect was rapidly reversed after drug removal. Conversely, nucleoside reverse transcriptase inhibitor zidovudine had no effect. Furthermore, in vivo ritonavir administration to adult mice reduced frontal cortex myelin protein levels. Finally, prefrontal cortex tissue from HIV-positive individuals with HAND on cART showed a significant decrease in myelin basic protein compared with untreated HIV-positive individuals with HAND or HIV-negative controls. These findings demonstrate that antiretrovirals can impact myelin integrity and have implications for myelination in juvenile HIV patients and myelin maintenance in adults on lifelong therapy.

Antiretroviral drugs induce oxidative stress and neuronal damage in the central nervous system.

HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery. Of note, potential toxicity of antiretroviral drugs in the central nervous system (CNS) remains remarkably underexplored and may contribute to the persistence of HAND in the cART era. Previous studies have shown antiretrovirals (ARVs) to be neurotoxic in the peripheral nervous system in vivo and in peripheral neurons in vitro. Alterations in lipid and protein metabolism, mitochondrial damage, and oxidative stress all play a role in peripheral ARV neurotoxicity. We hypothesized that ARVs also induce cellular stresses in the CNS, ultimately leading to neuronal damage and contributing to the changing clinical and pathological picture seen in HIV-positive patients in the cART era. In this report, we show that ARVs are neurotoxic in the CNS in both pigtail macaques and rats in vivo. Furthermore, in vitro, ARVs lead to accumulation of reactive oxygen species (ROS), and ultimately induction of neuronal damage and death. Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death. These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.