SARS-CoV-2 brainstem encephalitis in human inherited DBR1 deficiency.

Inherited deficiency of the RNA lariat-debranching enzyme 1 (DBR1) is a rare etiology of brainstem viral encephalitis. The cellular basis of disease and the range of viral predisposition are unclear. We report inherited DBR1 deficiency in a 14-year-old boy who suffered from isolated SARS-CoV-2 brainstem encephalitis. The patient is homozygous for a previously reported hypomorphic and pathogenic DBR1 variant (I120T). Consistently, DBR1 I120T/I120T fibroblasts from affected individuals from this and another unrelated kindred have similarly low levels of DBR1 protein and high levels of RNA lariats. DBR1 I120T/I120T human pluripotent stem cell (hPSC)-derived hindbrain neurons are highly susceptible to SARS-CoV-2 infection. Exogenous WT DBR1 expression in DBR1 I120T/I120T fibroblasts and hindbrain neurons rescued the RNA lariat accumulation phenotype. Moreover, expression of exogenous RNA lariats, mimicking DBR1 deficiency, increased the susceptibility of WT hindbrain neurons to SARS-CoV-2 infection. Inborn errors of DBR1 impair hindbrain neuron-intrinsic antiviral immunity, predisposing to viral infections of the brainstem, including that by SARS-CoV-2.

Encephalitis and poor neuronal death-mediated control of herpes simplex virus in human inherited RIPK3 deficiency.

Inborn errors of TLR3-dependent type I IFN immunity in cortical neurons underlie forebrain herpes simplex virus-1 (HSV-1) encephalitis (HSE) due to uncontrolled viral growth and subsequent cell death. We report an otherwise healthy patient with HSE who was compound heterozygous for nonsense (R422*) and frameshift (P493fs9*) RIPK3 variants. Receptor-interacting protein kinase 3 (RIPK3) is a ubiquitous cytoplasmic kinase regulating cell death outcomes, including apoptosis and necroptosis. In vitro, the R422* and P493fs9* RIPK3 proteins impaired cellular apoptosis and necroptosis upon TLR3, TLR4, or TNFR1 stimulation and ZBP1/DAI-mediated necroptotic cell death after HSV-1 infection. The patient's fibroblasts displayed no detectable RIPK3 expression. After TNFR1 or TLR3 stimulation, the patient's cells did not undergo apoptosis or necroptosis. After HSV-1 infection, the cells supported excessive viral growth despite normal induction of antiviral IFN-ß and IFN-stimulated genes (ISGs). This phenotype was, nevertheless, rescued by application of exogenous type I IFN. The patient's human pluripotent stem cell (hPSC)-derived cortical neurons displayed impaired cell death and enhanced viral growth after HSV-1 infection, as did isogenic RIPK3-knockout hPSC-derived cortical neurons. Inherited RIPK3 deficiency therefore confers a predisposition to HSE by impairing the cell death-dependent control of HSV-1 in cortical neurons but not their production of or response to type I IFNs.

Roles of the multivalent dynein adaptors BicD2 and RILP in neurons.

Cytoplasmic dynein is responsible for all forms of retrograde transport in neurons and other cells. Work over several years has led to the identification of a class of coiled-coil domain containing "adaptor" proteins that are responsible for expanding dynein's range of cargo interactions, as well as regulating dynein motor behavior. This brief review focuses first on the BicD family of adaptor proteins, which clearly serve to expand the number of dynein cargo interactions. RILP, another adaptor protein, also interacts with multiple proteins. Surprisingly, this is to mediate a series of steps within a common pathway, higher eukaryotic autophagy. These distinct features have important implications for understanding the full range of dynein adaptor functions.

A RILP-regulated pathway coordinating autophagosome biogenesis with transport.

Mammalian cells, including neurons, use macroautophagy (here 'autophagy') to degrade damaged proteins and organelles, and recycle nutrients in response to starvation and other forms of cell stress. The basic cellular machinery responsible for autophagy is highly conserved from yeast to mammals. However, evidence for specific adaptations to more complex organisms and in highly differentiated cells (e. g. neurons) remains limited. RILP (Rab interacting lysosomal protein) mediates retrograde transport of late endosomes (LEs) in nonneuronal mammalian cells. We have now found that RILP plays additional important, fundamental roles in neuronal autophagosome (AP) transport, and, more surprisingly, in AP biogenesis, and cargo turnover as well. RILP accomplishes these tasks via sequential interactions with key autophagosomal components - ATG5 and LC3 - as well as the microtubule motor protein cytoplasmic dynein (Figure 1A). We found further that RILP expression and behavior are controlled by MTOR kinase, linking RILP to a potentially wide range of physiological and pathophysiological functions.

The Dynein Adaptor RILP Controls Neuronal Autophagosome Biogenesis, Transport, and Clearance.

Autophagy plays critical roles in neurodegeneration and development, but how this pathway is organized and regulated in neurons remains poorly understood. Here, we find that the dynein adaptor RILP is essential for retrograde transport of neuronal autophagosomes, and surprisingly, their biogenesis as well. We find that induction of autophagy by mTOR inhibition specifically upregulates RILP expression and its localization to autophagosomes. RILP depletion or mutations in its LC3-binding LIR motifs strongly decrease autophagosome numbers suggesting an unexpected RILP role in autophagosome biogenesis. We find that RILP also interacts with ATG5 on isolation membranes, precluding premature dynein recruitment and autophagosome transport. RILP inhibition impedes autophagic turnover and causes p62/sequestosome-1 aggregation. Together, our results identify an mTOR-responsive neuronal autophagy pathway, wherein RILP integrates the processes of autophagosome biogenesis and retrograde transport to control autophagic turnover. This pathway has important implications for understanding how autophagy contributes to neuronal function, development, and disease.

Imaging of motor-dependent transport in neuronal and nonneuronal cells at high spatial and temporal resolution.

A wide range of subcellular organelles, pathogens, and macromolecular complexes are actively transported within neuronal and nonneuronal cells by microtubule motors. Transport speeds range up to 2-3 µm/s, which requires millisecond- and nanometer-scale resolution for proper imaging and analysis. Dissecting the contributions of multiple motor types has been challenging because of their functional interdependence and the complexity of individual motor behavior. In this chapter, we describe several methods for motor inhibition coupled with high-resolution particle tracking of vesicular and virus cargoes to provide a detailed and quantitative understanding of motor behavior and regulation. We discuss long-term inhibition, as well as short-term inhibition methods when needed to minimize complications from motor protein interactions.