Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calciumsensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.CNS trauma | live imaging | calcium influx | autophagy A xonal degeneration plays a pivotal role in the pathogenesis of numerous neurological disorders frequently preceding neuronal cell death and resulting in persistent functional disability. Traumatic spinal cord or peripheral nerve injury represent classical conditions where mechanical disruption of axonal integrity results in nervous system dysfunction (1, 2). Several degenerative CNS diseases show prominent axonal pathology already early in the disease course, such as the degeneration of nigrostriatal projection tracts or cardiac sympathetic nerves in Parkinson's disease (3) or corticospinal tracts in amyotrophic lateral sclerosis (4). Key features of axonal degeneration seem to be similar despite variable etiology. The distal part of the lesioned axon undergoes Wallerian degeneration (WD) characterized by initial axonal stability followed by rapid degeneration, fragmentation, and blebbing of the remaining axon, microtubule disassembly, and phagocytic clearance of the lesion site. The proximal part was reported to remain more stable than its distal counterpart (5-8), but imaging of the spinal cord in vivo visualized mechanisms of acute axonal degeneration (AAD) within the first minutes after lesion. In contrast to WD, AAD results in sudden axonal disintegration and extended for ≈300 μm proximal and distal to the lesion (9). One of the putative initiating steps in axonal degeneration is the influx of extracellular calcium, which is suggested to destabilize the axon and to transmit apoptotic signals to the neuronal soma (10-12).The optic nerve (ON) represents a unique model system for the study of axonal pathology in the CNS because of its accessibility and the possibility to manipulate the system...

Excitatory and inhibitory balance of neuronal network activity is essential for normal brain function and may be of particular importance to memory. Apolipoprotein (apo) E4 and amyloid-␤ (A␤) peptides, two major players in Alzheimer's disease (AD), cause inhibitory interneuron impairments and aberrant neuronal activity in the hippocampal dentate gyrus in AD-related mouse models and humans, leading to learning and memory deficits. To determine whether replacing the lost or impaired interneurons rescues neuronal signaling and behavioral deficits, we transplanted embryonic interneuron progenitors into the hippocampal hilus of aged apoE4 knock-in mice without or with A␤ accumulation. In both conditions, the transplanted cells developed into mature interneurons, functionally integrated into the hippocampal circuitry, and restored normal learning and memory. Thus, restricted hilar transplantation of inhibitory interneurons restores normal cognitive function in two widely used AD-related mouse models, highlighting the importance of interneuron impairments in AD pathogenesis and the potential of cell replacement therapy for AD. More broadly, it demonstrates that excitatory and inhibitory balance are crucial for learning and memory, and suggests an avenue for investigating the processes of learning and memory and their alterations in healthy aging and diseases.

SummaryTauopathies represent a group of neurodegenerative disorders characterized by the accumulation of pathological TAU protein in brains. We report a human neuronal model of tauopathy derived from induced pluripotent stem cells (iPSCs) carrying a TAU-A152T mutation. Using zinc-finger nuclease-mediated gene editing, we generated two isogenic iPSC lines: one with the mutation corrected, and another with the homozygous mutation engineered. The A152T mutation increased TAU fragmentation and phosphorylation, leading to neurodegeneration and especially axonal degeneration. These cellular phenotypes were consistent with those observed in a patient with TAU-A152T. Upon mutation correction, normal neuronal and axonal morphologies were restored, accompanied by decreases in TAU fragmentation and phosphorylation, whereas the severity of tauopathy was intensified in neurons with the homozygous mutation. These isogenic TAU-iPSC lines represent a critical advancement toward the accurate modeling and mechanistic study of tauopathies with human neurons and will be invaluable for drug-screening efforts and future cell-based therapies.

Apolipoprotein (apo) E4 is expressed in many types of brain cells, is associated with age-dependent decline of learning and memory in humans, and is the major genetic risk factor for AD. To determine whether the detrimental effects of apoE4 depend on its cellular sources, we generated human apoE knock-in mouse models in which the human APOE gene is conditionally deleted in astrocytes, neurons, or GABAergic interneurons. Here we report that deletion of apoE4 in astrocytes does not protect aged mice from apoE4-induced GABAergic interneuron loss and learning and memory deficits. In contrast, deletion of apoE4 in neurons does protect aged mice from both deficits. Furthermore, deletion of apoE4 in GABAergic interneurons is sufficient to gain similar protection. This study demonstrates a detrimental effect of endogenously produced apoE4 on GABAergic interneurons that leads to learning and memory deficits in mice and provides a novel target for drug development for AD related to apoE4.