Bohumil Maco

Increased axonal bouton dynamics in the aging mouse cortex

Significance Synaptic plasticity is considered an essential process for the formation and maintenance of memory. It had been assumed for decades that cognitive deficits within the aging brain result from reduced synaptic density and plasticity. By imaging axonal arbors and boutons in the aged brain, we surprisingly find the opposite, i.e., dramatically increased rates of synapse formation, elimination, and destabilization in specific cortical circuits. This observation suggests that learning and memory deficits in the aged brain may arise not through an inability to form new synapses but rather through decreased synaptic tenacity. Aging is a major risk factor for many neurological diseases and is associated with mild cognitive decline. Previous studies suggest that aging is accompanied by reduced synapse number and synaptic plasticity in specific brain regions. However, most studies, to date, used either postmortem or ex vivo preparations and lacked key in vivo evidence. Thus, whether neuronal arbors and synaptic structures remain dynamic in the intact aged brain and whether specific synaptic deficits arise during aging remains unknown. Here we used in vivo two-photon imaging and a unique analysis method to rigorously measure and track the size and location of axonal boutons in aged mice. Unexpectedly, the aged cortex shows circuit-specific increased rates of axonal bouton formation, elimination, and destabilization. Compared with the young adult brain, large (i.e., strong) boutons show 10-fold higher rates of destabilization and 20-fold higher turnover in the aged cortex. Size fluctuations of persistent boutons, believed to encode long-term memories, also are larger in the aged brain, whereas bouton size and density are not affected. Our data uncover a striking and unexpected increase in axonal bouton dynamics in the aged cortex. The increased turnover and destabilization rates of large boutons indicate that learning and memory deficits in the aged brain arise not through an inability to form new synapses but rather through decreased synaptic tenacity. Overall our study suggests that increased synaptic structural dynamics in specific cortical circuits may be a mechanism for age-related cognitive decline.

The Relationship between PSD-95 Clustering and Spine Stability In Vivo

The appearance and disappearance of dendritic spines, accompanied by synapse formation and elimination may underlie the experience-dependent reorganization of cortical circuits. The exact temporal relationship between spine and synapse formation in vivo remains unclear, as does the extent to which synapse formation enhances the stability of newly formed spines and whether transient spines produce synapses. We used in utero electroporation of DsRedExpress- and eGFP-tagged postsynaptic density protein 95 (PSD-95) to investigate the relationship between spine and PSD stability in mouse neocortical L2/3 pyramidal cells in vivo. Similar to previous studies, spines and synapses appeared and disappeared, even in naive animals. Cytosolic spine volumes and PSD-95-eGFP levels in spines covaried over time, suggesting that the strength of many individual synapses continuously changes in the adult neocortex. The minority of newly formed spines acquired PSD-95-eGFP puncta. Spines that failed to acquire a PSD rarely survived for more than a day. Although PSD-95-eGFP accumulation was associated with increased spine lifetimes, most new spines with a PSD did not convert into persistent spines. This indicates that transient spines may serve to produce short-lived synaptic contacts. Persistent spines that were destined to disappear showed, on average, reduced PSD-95-eGFP levels well before the actual pruning event. Altogether, our data indicate that the PSD size relates to spine stability in vivo.

Altered Synaptic Dynamics during Normal Brain Aging

What is the neuroanatomical basis for the decline in brain function that occurs during normal aging? Previous postmortem studies have blamed it on a reduction in spine density, though results remain controversial and spine dynamics were not assessed. We used chronic in vivo two-photon imaging of dendritic spines and axonal boutons in somatosensory cortex for up to 1 year in thy1 GFP mice to test the hypothesis that aging is associated with alterations in synaptic dynamics. We find that the density of spines and en passant boutons (EPBs) in pyramidal cells increases throughout adult life but is stable between mature (8–15 months) and old (>20 months) mice. However, new spines and EPBs are two to three times more likely to be stabilized over 30 d in old mice, although the long-term retention (over months) of stable spines is lower in old animals. In old mice, spines are smaller on average but are still able to make synaptic connections regardless of their size, as assessed by serial section electron microscopy reconstructions of previously imaged dendrites. Thus, our data suggest that age-related deficits in sensory perception are not associated with synapse loss in somatosensory cortex (as might be expected) but with alterations in the size and stability of spines and boutons observed in this brain area. The changes we describe here likely result in weaker synapses that are less capable of short-term plasticity in aged individuals, and therefore to less efficient circuits.

In vivo single branch axotomy induces GAP-43–dependent sprouting and synaptic remodeling in cerebellar cortex

Plasticity in the central nervous system in response to injury is a complex process involving axonal remodeling regulated by specific molecular pathways. Here, we dissected the role of growth-associated protein 43 (GAP-43; also known as neuromodulin and B-50) in axonal structural plasticity by using, as a model, climbing fibers. Single axonal branches were dissected by laser axotomy, avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Despite the very small denervated area, the injured axons consistently reshape the connectivity with surrounding neurons. At the same time, adult climbing fibers react by sprouting new branches through the intact surroundings. Newly formed branches presented varicosities, suggesting that new axons were more than just exploratory sprouts. Correlative light and electron microscopy reveals that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites. By using an RNA interference approach, we found that downregulating GAP-43 causes a significant increase in the turnover of presynaptic boutons. In addition, silencing hampers the generation of reactive sprouts. Our findings show the requirement of GAP-43 in sustaining synaptic stability and promoting the initiation of axonal regrowth.

Correlative In Vivo 2 Photon and Focused Ion Beam Scanning Electron Microscopy of Cortical Neurons

Correlating in vivo imaging of neurons and their synaptic connections with electron microscopy combines dynamic and ultrastructural information. Here we describe a semi-automated technique whereby volumes of brain tissue containing axons and dendrites, previously studied in vivo, are subsequently imaged in three dimensions with focused ion beam scanning electron microcopy. These neurites are then identified and reconstructed automatically from the image series using the latest segmentation algorithms. The fast and reliable imaging and reconstruction technique avoids any specific labeling to identify the features of interest in the electron microscope, and optimises their preservation and staining for 3D analysis.

In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits

To what extent, how and when axons respond to injury in the highly interconnected grey matter is poorly understood. Here we use two-photon imaging and focused ion beam-scanning electron microscopy to explore, at synaptic resolution, the regrowth capacity of several neuronal populations in the intact brain. Time-lapse analysis of >100 individually ablated axons for periods of up to a year reveals a surprising inability to regenerate even in a glial scar-free environment. However, depending on cell type some axons spontaneously extend for distances unseen in the unlesioned adult cortex and at maximum speeds comparable to peripheral nerve regeneration. Regrowth follows a distinct pattern from developmental axon growth. Remarkably, although never reconnecting to the original targets, axons consistently form new boutons at comparable prelesion synaptic densities, implying the existence of intrinsic homeostatic programmes, which regulate synaptic numbers on regenerating axons. Our results may help guide future clinical investigations to promote functional axon regeneration.

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

The role of extracellular α-synuclein (α-syn) in the initiation and the spreading of neurodegeneration in Parkinson’s disease (PD) has been studied extensively over the past 10 years. However, the nature of the α-syn toxic species and the molecular mechanisms by which they may contribute to neuronal cell loss remain controversial. In this study, we show that fully characterized recombinant monomeric, fibrillar or stabilized forms of oligomeric α-syn do not trigger significant cell death when added individually to neuroblastoma cell lines. However, a mixture of preformed fibrils (PFFs) with monomeric α-syn becomes toxic under conditions that promote their growth and amyloid formation. In hippocampal primary neurons and ex vivo hippocampal slice cultures, α-syn PFFs are capable of inducing a moderate toxicity over time that is greatly exacerbated upon promoting fibril growth by addition of monomeric α-syn. The causal relationship between α-syn aggregation and cellular toxicity was further investigated by assessing the effect of inhibiting fibrillization on α-syn-induced cell death. Remarkably, our data show that blocking fibril growth by treatment with known pharmacological inhibitor of α-syn fibrillization (Tolcapone) or replacing monomeric α-syn by monomeric β-synuclein in α-syn mixture composition prevent α-syn-induced toxicity in both neuroblastoma cell lines and hippocampal primary neurons. We demonstrate that exogenously added α-syn fibrils bind to the plasma membrane and serve as nucleation sites for the formation of α-syn fibrils and promote the accumulation and internalization of these aggregates that in turn activate both the extrinsic and intrinsic apoptotic cell death pathways in our cellular models. Our results support the hypothesis that ongoing aggregation and fibrillization of extracellular α-syn play central roles in α-syn extracellular toxicity, and suggest that inhibiting fibril growth and seeding capacity constitute a viable strategy for protecting against α-syn-induced toxicity and slowing the progression of neurodegeneration in PD and other synucleinopathies.

Semiautomated correlative 3D electron microscopy of in vivo–imaged axons and dendrites

This protocol describes how in vivo–imaged dendrites and axons in adult mouse brains can subsequently be prepared and imaged with focused ion beam scanning electron microscopy (FIBSEM). The procedure starts after in vivo imaging with chemical fixation, followed by the identification of the fluorescent structures of interest. Their position is then highlighted in the fixed tissue by burning fiducial marks with the two-photon laser. Once the section has been stained and resin-embedded, a small block is trimmed close to these marks. Serially aligned EM images are acquired through this region, using FIBSEM, and the neurites of interest are then reconstructed semiautomatically by using the ilastik software (http://ilastik.org/). This reliable imaging and reconstruction technique avoids the use of specific labels to identify the structures of interest in the electron microscope, enabling optimal chemical fixation techniques to be applied and providing the best possible structural preservation for 3D analysis. The entire protocol takes ∼4 d.

Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

Significance Although it has been established for over 100 years that Lewy bodies (LBs) represent the major pathological hallmark of Parkinsons disease (PD), we still do not know why these fibrillar intraneuronal inclusions of α-synuclein (α-Syn) protein form, or how they contribute to disease progression. One of the major causes underlying this gap in knowledge is the lack of animal models that reproduce the formation of fibrillar LB-like inclusions. In this study, we show that the absence of human α-Syn (hα-Syn) fibrillization into LBs in mice can be attributed to interactions between hα-Syn and its endogenously expressed mouse α-Syn homologue. Moreover, we provide well-characterized primary neuronal and in vivo models that recapitulate the main molecular feature of PD, bona fide α-Syn fibrillization. Lewy bodies (LBs) are intraneuronal inclusions consisting primarily of fibrillized human α-synuclein (hα-Syn) protein, which represent the major pathological hallmark of Parkinsons disease (PD). Although doubling hα-Syn expression provokes LB pathology in humans, hα-Syn overexpression does not trigger the formation of fibrillar LB-like inclusions in mice. We hypothesized that interactions between exogenous hα-Syn and endogenous mouse synuclein homologs could be attenuating hα-Syn fibrillization in mice, and therefore, we systematically assessed hα-Syn aggregation propensity in neurons derived from α-Syn–KO, β-Syn–KO, γ-Syn–KO, and triple-KO mice lacking expression of all three synuclein homologs. Herein, we show that hα-Syn forms hyperphosphorylated (at S129) and ubiquitin-positive LB-like inclusions in primary neurons of α-Syn–KO, β-Syn–KO, and triple-KO mice, as well as in transgenic α-Syn–KO mouse brains in vivo. Importantly, correlative light and electron microscopy, immunogold labeling, and thioflavin-S binding established their fibrillar ultrastructure, and fluorescence recovery after photobleaching/photoconversion experiments showed that these inclusions grow in size and incorporate soluble proteins. We further investigated whether the presence of homologous α-Syn species would interfere with the seeding and spreading of α-Syn pathology. Our results are in line with increasing evidence demonstrating that the spreading of α-Syn pathology is most prominent when the injected preformed fibrils and host-expressed α-Syn monomers are from the same species. These findings provide insights that will help advance the development of neuronal and in vivo models for understanding mechanisms underlying hα-Syn intraneuronal fibrillization and its contribution to PD pathogenesis, and for screening pharmacologic and genetic modulators of α-Syn fibrillization in neurons.

Correlative In Vivo 2-Photon Imaging and Focused Ion Beam Scanning Electron Microscopy: 3D Analysis of Neuronal Ultrastructure

This protocol describes how dendrites and axons, imaged in vivo, can subsequently be analyzed in 3D using focused ion beam scanning electron microscopy (FIBSEM). The fluorescent structures are identified after chemical fixation and their position highlighted using the 2-photon laser to burn fiducial marks around the region. Once the section has been stained and resin embedded, a small block is trimmed close to these marks. Serially aligned EM images are acquired through this region, using FIBSEM, and the neurites of interest then reconstructed semi-automatically using the Ilastik software (ilastik.org). This fast and reliable imaging and reconstruction technique avoids the use of specific labels to identify the features of interest in the electron microscope and optimizes their preservation for high-quality imaging and 3D analysis.