Synapsin E-domain is essential for α-synuclein function.

The cytosolic proteins synucleins and synapsins are thought to play cooperative roles in regulating synaptic vesicle (SV) recycling, but mechanistic insight is lacking. Here we identify the synapsin E-domain as an essential functional binding-partner of α-synuclein (α-syn). Synapsin E-domain allows α-syn functionality, binds to α-syn, and is necessary and sufficient for enabling effects of α-syn at the synapse. Together with previous studies implicating the E-domain in clustering SVs, our experiments advocate a cooperative role for these two proteins in maintaining physiologic SV clusters.

Diminished Neuronal ESCRT-0 Function Exacerbates AMPA Receptor Derangement and Accelerates Prion-Induced Neurodegeneration.

Endolysosomal defects in neurons are central to the pathogenesis of prion and other neurodegenerative disorders. In prion disease, prion oligomers traffic through the multivesicular body (MVB) and are routed for degradation in lysosomes or for release in exosomes, yet how prions impact proteostatic pathways is unclear. We found that prion-affected human and mouse brain showed a marked reduction in Hrs and STAM1 (ESCRT-0), which route ubiquitinated membrane proteins from early endosomes into MVBs. To determine how the reduction in ESCRT-0 impacts prion conversion and cellular toxicity in vivo, we prion-challenged conditional knockout mice (male and female) having Hrs deleted from neurons, astrocytes, or microglia. The neuronal, but not astrocytic or microglial, Hrs-depleted mice showed a shortened survival and an acceleration in synaptic derangements, including an accumulation of ubiquitinated proteins, deregulation of phosphorylated AMPA and metabotropic glutamate receptors, and profoundly altered synaptic structure, all of which occurred later in the prion-infected control mice. Finally, we found that neuronal Hrs (nHrs) depletion increased surface levels of the cellular prion protein, PrPC, which may contribute to the rapidly advancing disease through neurotoxic signaling. Taken together, the reduced Hrs in the prion-affected brain hampers ubiquitinated protein clearance at the synapse, exacerbates postsynaptic glutamate receptor deregulation, and accelerates neurodegeneration.SIGNIFICANCE STATEMENT Prion diseases are rapidly progressive neurodegenerative disorders characterized by prion aggregate spread through the central nervous system. Early disease features include ubiquitinated protein accumulation and synapse loss. Here, we investigate how prion aggregates alter ubiquitinated protein clearance pathways (ESCRT) in mouse and human prion-infected brain, discovering a marked reduction in Hrs. Using a prion-infection mouse model with neuronal Hrs (nHrs) depleted, we show that low neuronal Hrs is detrimental and markedly shortens survival time while accelerating synaptic derangements, including ubiquitinated protein accumulation, indicating that Hrs loss exacerbates prion disease progression. Additionally, Hrs depletion increases the surface distribution of prion protein (PrPC), linked to aggregate-induced neurotoxic signaling, suggesting that Hrs loss in prion disease accelerates disease through enhancing PrPC-mediated neurotoxic signaling.

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins.

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the healthy mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo.

Imaging Diversity in Slow Axonal Transport.

The polarized morphology of neurons necessitates the delivery of proteins synthesized in the soma along the length of the axon to distal synapses; critical for sustaining communication between neurons. This constitutive and dynamic process of protein transport along axons termed "axonal transport" was initially characterized by classic pulse-chase radiolabeling studies which identified two major rate components: a fast component and a slow component. Early radiolabeling studies indicated "cohesive co-transport" of slow transport cargos. However, this approach could not be used to visualize or provide mechanistic insights on this highly dynamic process. The advent of fluorescent and photoactivatable imaging probes have now enabled real-time imaging of axonal transport. Conventional fluorescent probes have helped visualize and characterize the molecular mechanisms of transport of vesicular proteins. These proteins typically move in the fast component of axonal transport and appear as "punctate structures" along axons. However, a large majority of transported proteins that move in the slow component of transport, typically show a "uniform diffusive glow" along axons when tagged to conventional fluorescent probes. This makes it challenging to unequivocally track them in real time. Our lab has used photoactivatable fluorescent probes to tag three individual cytosolic proteins moving in the slow component of axonal transport, and identified three distinct modes of transport along axons. Our data from these experiments argue against the prevailing hypothesis based on classic radiolabeling studies, which suggested that all slow-transport proteins may move along the axon as one large macromolecular protein complex. Although other labs have started using photoactivation to study axonal transport of cytosolic proteins, this technique remains largely under-utilized. Here, we describe the detailed protocols to image and analyze axonal transport of three typical slow-component cargoes along axons of cultured hippocampal neurons.

Dopamine neurons exhibit emergent glutamatergic identity in Parkinson's disease.

Loss of midbrain dopamine neurons causes the cardinal symptoms of Parkinson's disease. However, not all dopamine neurons are equally vulnerable and a better understanding of the cell-type specific properties relating to selective dopamine neuron degeneration is needed. Most midbrain dopamine neurons express the vesicular glutamate transporter VGLUT2 during development and a subset continue to express low levels of VGLUT2 in adulthood, enabling the co-release of glutamate. Moreover, VGLUT2 expression in dopamine neurons can be neuroprotective since its genetic disruption was shown to sensitize dopamine neurons to neurotoxins. Here, we show that in response to toxic insult, and in two distinct models of alpha-synuclein stress, VGLUT2 dopamine neurons were resilient to degeneration. Dopamine neurons expressing VGLUT2 were enriched whether or not insult induced dopamine neuron loss, suggesting that while VGLUT2 dopamine neurons are more resilient, VGLUT2 expression can also be transcriptionally upregulated by injury. Finally, we observed that VGLUT2 expression was enhanced in surviving dopamine neurons from post-mortem Parkinson's disease individuals. These data indicate that emergence of a glutamatergic identity in dopamine neurons may be part of a neuroprotective response in Parkinson's disease.

Clathrin packets move in slow axonal transport and deliver functional payloads to synapses.

In non-neuronal cells, clathrin has established roles in endocytosis, with clathrin cages enclosing plasma membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin is conveyed in slow axonal transport over days to weeks, and the underlying transport/targeting mechanisms, mobile cargo structures, and even its precise presynaptic localization and physiologic role are unclear. Combining live imaging, photobleaching/conversion, mass spectrometry, electron microscopy, and super-resolution imaging, we found that unlike in dendrites, where clathrin cages rapidly assemble and disassemble, in axons, clathrin and related proteins organize into stable "transport packets" that are unrelated to endocytosis and move intermittently on microtubules, generating an overall slow anterograde flow. At synapses, multiple clathrin packets abut synaptic vesicle (SV) clusters, and clathrin packets also exchange between synaptic boutons in a microtubule-dependent "superpool." Within synaptic boundaries, clathrin is surprisingly dynamic, continuously exchanging between local clathrin assemblies, and its depletion impairs SV recycling. Our data provide a conceptual framework for understanding clathrin trafficking and presynaptic targeting that has functional implications.

Gene-based therapies for neurodegenerative diseases.

Gene therapy is making a comeback. With its twin promise of targeting disease etiology and 'long-term correction', gene-based therapies (defined here as all forms of genome manipulation) are particularly appealing for neurodegenerative diseases, for which conventional pharmacologic approaches have been largely disappointing. The recent success of a viral-vector-based gene therapy in spinal muscular atrophy-promoting survival and motor function with a single intravenous injection-offers a paradigm for such therapeutic intervention and a platform to build on. Although challenges remain, the newfound optimism largely stems from advances in the development of viral vectors that can diffusely deliver genes throughout the CNS, as well as genome-engineering tools that can manipulate disease pathways in ways that were previously impossible. Surely spinal muscular atrophy cannot be the only neurodegenerative disease amenable to gene therapy, and one can imagine a future in which the toolkit of a clinician will include gene-based therapeutics. The goal of this Review is to highlight advances in the development and application of gene-based therapies for neurodegenerative diseases and offer a prospective look into this emerging arena.

Finding order in slow axonal transport.

Slow axonal transport conveys cytosolic and cytoskeletal proteins into axons and synapses at overall velocities that are several orders of magnitude slower than the fast transport of membranous organelles such as vesicles and mitochondria. The phenomenon of slow transport was characterized by in vivo pulse-chase radiolabeling studies done decades ago, and proposed models emphasized an orderly cargo-movement, with apparent cohesive transport of multiple proteins and subcellular structures along axons over weeks to months. However, visualization of cytosolic and cytoskeletal cargoes in cultured neurons at much higher temporal and spatial resolution has revealed an unexpected diversity in movement - ranging from a diffusion-like biased motion, to intermittent cargo dynamics and unusual polymerization-based transport paradigms. This review provides an updated view of slow axonal transport and explores emergent mechanistic themes in this enigmatic rate-class.

Synapsins regulate α-synuclein functions.

The normal function of α-synuclein (α-syn) remains elusive. Although recent studies suggest α-syn as a physiologic attenuator of synaptic vesicle (SV) recycling, mechanisms are unclear. Here, we show that synapsin-a cytosolic protein with known roles in SV mobilization and clustering-is required for presynaptic functions of α-syn. Our data offer a critical missing link and advocate a model where α-syn and synapsin cooperate to cluster SVs and attenuate recycling.

Functional cooperation of α-synuclein and VAMP2 in synaptic vesicle recycling.

The function of α-synuclein (α-syn) has been long debated, and two seemingly divergent views have emerged. In one, α-syn binds to VAMP2, acting as a SNARE chaperone-but with no effect on neurotransmission-while another posits that α-syn attenuates neurotransmitter release by restricting synaptic vesicle mobilization and recycling. Here, we show that α-syn-VAMP2 interactions are necessary for α-syn-induced synaptic attenuation. Our data connect divergent views and suggest a unified model of α-syn function.

Proteomic Atlas of the Human Brain in Alzheimer's Disease.

The brain represents one of the most divergent and critical organs in the human body. Yet, it can be afflicted by a variety of neurodegenerative diseases specifically linked to aging, about which we lack a full biomolecular understanding of onset and progression, such as Alzheimer's disease (AD). Here we provide a proteomic resource comprising nine anatomically distinct sections from three aged individuals, across a spectrum of disease progression, categorized by quantity of neurofibrillary tangles. Using state-of-the-art mass spectrometry, we identify a core brain proteome that exhibits only small variance in expression, accompanied by a group of proteins that are highly differentially expressed in individual sections and broader regions. AD affected tissue exhibited slightly elevated levels of tau protein with similar relative expression to factors associated with the AD pathology. Substantial differences were identified between previous proteomic studies of mature adult brains and our aged cohort. Our findings suggest considerable value in examining specifically the brain proteome of aged human populations from a multiregional perspective. This resource can serve as a guide, as well as a point of reference for how specific regions of the brain are affected by aging and neurodegeneration.

CRISPR/Cas9 editing of APP C-terminus attenuates ß-cleavage and promotes α-cleavage.

CRISPR/Cas9 guided gene-editing is a potential therapeutic tool, however application to neurodegenerative disease models has been limited. Moreover, conventional mutation correction by gene-editing would only be relevant for the small fraction of neurodegenerative cases that are inherited. Here we introduce a CRISPR/Cas9-based strategy in cell and animal models to edit endogenous amyloid precursor protein (APP) at the extreme C-terminus and reciprocally manipulate the amyloid pathway, attenuating APP-ß-cleavage and Aß production, while up-regulating neuroprotective APP-α-cleavage. APP N-terminus and compensatory APP-homologues remain intact, with no apparent effects on neurophysiology in vitro. Robust APP-editing is seen in human iPSC-derived neurons and mouse brains with no detectable off-target effects. Our strategy likely works by limiting APP and BACE-1 approximation, and we also delineate mechanistic events that abrogates APP/BACE-1 convergence in this setting. Our work offers conceptual proof for a selective APP silencing strategy.

Processive flow by biased polymerization mediates the slow axonal transport of actin.

Classic pulse-chase studies have shown that actin is conveyed in slow axonal transport, but the mechanistic basis for this movement is unknown. Recently, we reported that axonal actin was surprisingly dynamic, with focal assembly/disassembly events ("actin hotspots") and elongating polymers along the axon shaft ("actin trails"). Using a combination of live imaging, superresolution microscopy, and modeling, in this study, we explore how these dynamic structures can lead to processive transport of actin. We found relatively more actin trails elongated anterogradely as well as an overall slow, anterogradely biased flow of actin in axon shafts. Starting with first principles of monomer/filament assembly and incorporating imaging data, we generated a quantitative model simulating axonal hotspots and trails. Our simulations predict that the axonal actin dynamics indeed lead to a slow anterogradely biased flow of the population. Collectively, the data point to a surprising scenario where local assembly and biased polymerization generate the slow axonal transport of actin without involvement of microtubules (MTs) or MT-based motors. Mechanistically distinct from polymer sliding, this might be a general strategy to convey highly dynamic cytoskeletal cargoes.

Actin Assemblies in the Axon Shaft - some Open Questions.

The actin cytoskeleton in neurons plays critical roles in axonal growth and synaptic organization. Until recently, most studies on axonal actin were limited to terminal growth cones or synapses, whereas the organization of actin along the shaft of the axon was relatively ignored. However, experiments using super-resolution microscopy and live imaging have revealed previously unknown actin structures along the axonal shaft, such as periodic 'actin rings' circumferentially wrapping underneath the plasma membrane and dynamic actin pools deeper within the axon shaft (termed actin 'hotspots' and 'trails'). In this short review, we highlight some open questions that have surfaced as a direct result of these discoveries.

The physical approximation of APP and BACE-1: A key event in alzheimer's disease pathogenesis.

Alzheimer's disease (AD) is characterized by the accumulation of insoluble deposits of Amyloid ß (Aß) in brains. Aß is derived by sequential cleavage of the amyloid precursor protein (APP) by ß-site secretase enzyme (BACE-1) and γ-secretase. Proteolytic processing of APP by BACE-1 is the rate-limiting step in Aß production, and this pathway is a prime target for AD drug development. Both APP and BACE-1 are membrane-spanning proteins, transported via secretory and endocytic pathways; and the physical interaction of APP and BACE-1 during trafficking is a key cell biological event initiating the amyloidogenic pathway. Here, we highlight recent research on intracellular trafficking/sorting of APP and BACE-1, and discuss how dysregulation of these pathways might lead to enhanced convergence of APP and BACE-1, and subsequent ß-cleavage of APP. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 340-347, 2018.

Author Correction: Synuclein and dopamine: the Bonnie and Clyde of Parkinson's disease.

In the version of this article initially published, the molecular mass of synuclein was given as 140 kDa instead of ~14 kDa. The error has been corrected in the HTML and PDF versions of the article.

The nano-architecture of the axonal cytoskeleton.

The corporeal beauty of the neuronal cytoskeleton has captured the imagination of generations of scientists. One of the easiest cellular structures to visualize by light microscopy, its existence has been known for well over 100 years, yet we have only recently begun to fully appreciate its intricacy and diversity. Recent studies combining new probes with super-resolution microscopy and live imaging have revealed surprising details about the axonal cytoskeleton and, in particular, have discovered previously unknown actin-based structures. Along with traditional electron microscopy, these newer techniques offer a nanoscale view of the axonal cytoskeleton, which is important for our understanding of neuronal form and function, and lay the foundation for future studies. In this Review, we summarize existing concepts in the field and highlight contemporary discoveries that have fundamentally altered our perception of the axonal cytoskeleton.

Hsc70 chaperone activity is required for the cytosolic slow axonal transport of synapsin.

Soluble cytosolic proteins vital to axonal and presynaptic function are synthesized in the neuronal soma and conveyed via slow axonal transport. Our previous studies suggest that the overall slow transport of synapsin is mediated by dynamic assembly/disassembly of cargo complexes followed by short-range vectorial transit (the "dynamic recruitment" model). However, neither the composition of these complexes nor the mechanistic basis for the dynamic behavior is understood. In this study, we first examined putative cargo complexes associated with synapsin using coimmunoprecipitation and multidimensional protein identification technology mass spectrometry (MS). MS data indicate that synapsin is part of a multiprotein complex enriched in chaperones/cochaperones including Hsc70. Axonal synapsin-Hsc70 coclusters are also visualized by two-color superresolution microscopy. Inhibition of Hsc70 ATPase activity blocked the slow transport of synapsin, disrupted axonal synapsin organization, and attenuated Hsc70-synapsin associations, advocating a model where Hsc70 activity dynamically clusters cytosolic proteins into cargo complexes, allowing transport. Collectively, our study offers insight into the molecular organization of cytosolic transport complexes and identifies a novel regulator of slow transport.

Amyloid-ß Peptide Is Needed for cGMP-Induced Long-Term Potentiation and Memory.

High levels of amyloid-ß peptide (Aß) have been related to Alzheimer's disease pathogenesis. However, in the healthy brain, low physiologically relevant concentrations of Aß are necessary for long-term potentiation (LTP) and memory. Because cGMP plays a key role in these processes, here we investigated whether the cyclic nucleotide cGMP influences Aß levels and function during LTP and memory. We demonstrate that the increase of cGMP levels by the phosphodiesterase-5 inhibitors sildenafil and vardenafil induces a parallel release of Aß due to a change in the approximation of amyloid precursor protein (APP) and the ß-site APP cleaving enzyme 1. Moreover, electrophysiological and behavioral studies performed on animals of both sexes showed that blocking Aß function, by using anti-murine Aß antibodies or APP knock-out mice, prevents the cGMP-dependent enhancement of LTP and memory. Our data suggest that cGMP positively regulates Aß levels in the healthy brain which, in turn, boosts synaptic plasticity and memory.SIGNIFICANCE STATEMENT Amyloid-ß (Aß) is a key pathogenetic factor in Alzheimer's disease. However, low concentrations of endogenous Aß, mimicking levels of the peptide in the healthy brain, enhance hippocampal long-term potentiation (LTP) and memory. Because the second messenger cGMP exerts a central role in LTP mechanisms, here we studied whether cGMP affects Aß levels and function during LTP. We show that cGMP enhances Aß production by increasing the APP/BACE-1 convergence in endolysosomal compartments. Moreover, the cGMP-induced enhancement of LTP and memory was disrupted by blockade of Aß, suggesting that the physiological effect of the cyclic nucleotide on LTP and memory is dependent upon Aß.