The functional imaging of neuronal circuits of the central nervous system is crucial for phenotype screenings or investigations of defects in neurodegenerative disorders. Current techniques yield either low penetration depth, yield poor resolution, or are restricted by the age of the animals. Here, we present a novel ultramicroscopy protocol for fluorescence imaging and three-dimensional reconstruction in the central nervous system of adult mice. In combination with tracing as a functional assay for axonal transport, retrogradely labeled descending motor neurons were visualized with >4 mm penetration depth. The analysis of the motor cortex shortly before the onset of clinical prion disease revealed that >80% neurons have functional impairments in axonal transport. Our study provides evidence that prion disease is associated with severe axonal transport defects in the cortical motor neurons and suggests a novel mechanism for prion-mediated neurodegeneration.

In sterile neuroinflammation, a pathological role is proposed for microglia, whereas in viral encephalitis, their function is not entirely clear. Many viruses exploit the odorant system and enter the CNS via the olfactory bulb (OB). Upon intranasal vesicular stomatitis virus instillation, we show an accumulation of activated microglia and monocytes in the OB. Depletion of microglia during encephalitis results in enhanced virus spread and increased lethality. Activation, proliferation, and accumulation of microglia are regulated by type I IFN receptor signaling of neurons and astrocytes, but not of microglia. Morphological analysis of myeloid cells shows that type I IFN receptor signaling of neurons has a stronger impact on the activation of myeloid cells than of astrocytes. Thus, in the infected CNS, the cross talk among neurons, astrocytes, and microglia is critical for full microglia activation and protection from lethal encephalitis.

Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally -- after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease.

Recent studies have shown that tissue macrophages (MΦ) arise from embryonic progenitors of the yolk sac (YS) and fetal liver and colonize tissues before birth. Further studies have proposed that developmentally distinct tissue MΦ can be identified based on the differential expression of F4/80 and CD11b, but whether a characteristic transcriptional profile exists is largely unknown. Here, we took advantage of an inducible fate-mapping system that facilitated the identification of CD45(+)c-kit(-)CX3CR1(+)F4/80(+) (A2) progenitors of the YS as the source of F4/80(hi) but not CD11b(hi) MΦ. Large-scale transcriptional profiling of MΦ precursors from the YS stage to adulthood allowed for building computational models for F4/80(hi) tissue macrophages being direct descendants of A2 progenitors. We further identified a distinct molecular signature of F4/80(hi) and CD11b(hi) MΦ and found that Irf8 was vital for MΦ maturation. Our data provide new cellular and molecular insights into the origin and developmental pathways of tissue MΦ.

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8+ T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8+ T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen's encephalitis patients. In this devastating CD8+ T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8+ T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.