. A large-scale nanoscopy and biochemistry analysis of postsynaptic dendritic spines. Nat Neurosci. 2021 Jun 24; PubMed.

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  1. I applaud this study for several reasons. One is the elegant combination of fairly sophisticated techniques that are rarely coupled together, i.e., super-resolution microscopy, deep quantitative protein analysis, and large-scale automated anatomical analysis to address a very specific but high-content question comparing the protein composition of two distinct subtypes of dendritic spines.

    It is inherently interesting, and perhaps initially counterintuitive, that the more transient stubby spines have similar protein composition and copy number as the more structurally and functionally stable mushroom spines. More in line with expectations, perhaps, is that the mushroom spine protein composition associates more strongly with synaptic strength, and thus is more facile in terms of long-term plasticity changes.  

    In terms of Alzheimer's disease research, it would be enlightening to explore the extent to which this protein population is altered during the course of AD pathogenesis, and when anticipated changes in spine composition change in relation to cognitive changes, histopathology and amyloid/tau aggregation, and other known cellular signaling alterations such as calcium dyshomeostasis. 

    Of particular note is the analysis and discussion surrounding calcium buffering capacity within spines. In the conditions within this study, it was estimated that cytosolic calcium buffers, including calmodulin, are present in excess to buffer resting calcium and influx through plasma membrane channels.

    Interestingly, and with consequences for AD pathophysiology, ER calcium buffers such as calreticulin are in sufficient concentration to buffer ER calcium (orders of magnitude higher than resting cytosolic levels) in stubby spines but not in mushroom spines. In AD mouse models, ER-calcium release within mushroom spines can be three to 10 times higher than in non-AD mice, and thus can have broad consequences on synaptic structure, function, and plasticity (Goussakov et al., 2010, 2011; Chakroborty et al., 2019). 

    Under these markedly increased calcium-release conditions, it is unclear if the described composition of cytosolic buffers is sufficient to adequately restore calcium levels within physiological timeframes and given the presumed lower levels of ER buffers in mushroom spines. It is currently unresolved how the ER calcium stores are affected in AD.

    Thus, under these imbalanced conditions, this study adds a key structural and biochemical perspective on how altered calcium homeostasis within a specific spine class can contribute to synaptic deficits and thus memory-encoding defects in the pathophysiology of AD.   

    References:

    . NMDA-mediated Ca(2+) influx drives aberrant ryanodine receptor activation in dendrites of young Alzheimer's disease mice. J Neurosci. 2010 Sep 8;30(36):12128-37. PubMed.

    . Generation of dendritic Ca2+ oscillations as a consequence of altered ryanodine receptor function in AD neurons. Channels (Austin). 2011 Jan-Feb;5(1):9-13. PubMed.

    . Reduced presynaptic vesicle stores mediate cellular and network plasticity defects in an early-stage mouse model of Alzheimer's disease. Mol Neurodegener. 2019 Jan 22;14(1):7. PubMed.

    View all comments by Grace Stutzmann
  2. This is indeed an interesting and well-done study. From an Alzheimer’s disease perspective, it is interesting that APP is observed in spines in cultured neurons; this supports the work of my group 15 years back (Priller et al., 2006). 

    Tau on the other hand, and proteins claimed to be involved in tau function at the dendritic spine, like fyn, do not show up. This fits my expectations; I still don’t believe that tau has a physiologic function at dendritic spines.

    These observations are constructive to validate the quality of synaptosomal preparations, which should contain these proteins if done well. For example, based on this dataset, changes in the proteome of synaptosomal preparations in α-synuclein-overexpressing mice (Blumenstock et al., 2019) can now be further analyzed regarding whether changes occur at mushroom or stubby spines.

    Also, this dataset is fundamental for the comparison to synapses that get formed in human iPSC-derived neurons.

    References:

    . Synapse formation and function is modulated by the amyloid precursor protein. J Neurosci. 2006 Jul 5;26(27):7212-21. PubMed.

    . Early defects in translation elongation factor 1α levels at excitatory synapses in α-synucleinopathy. Acta Neuropathol. 2019 Dec;138(6):971-986. Epub 2019 Aug 26 PubMed.

    View all comments by Jochen Herms
  3. In this paper, using a combination of various methods, the average copy numbers and spatial distribution of more than 100 proteins in mushroom and stubby types of dendritic spines were calculated. The shape of these spines differs to a great extent. One type has a neck connecting it to the dendrite; the other appears to be just a membrane protrusion, so there is some difficulty outlining its border, i.e., where the dendrite ends and the spine starts.

    It is a bit counterintuitive that these spines appear to have similar spine surface and PSD area as determined with the help of EM microscopy. Also, protein distribution and localization is not exhibiting big differences in morphological zones of spines. The authors found that the mushroom spines' machinery of trafficking and secretion is correlated to PSD area to a greater extent than for stubby spines, so the assumption was made that mushroom spines respond to changes in synaptic transmission more easily. 

    The authors mention that stubby spines are abundant during neuronal development, but mostly disappear in mature neurons. Is the question still if these stubby spines analyzed in adult neurons are similar to stubby spines in developing neurons? Because there is a possibility that stubby spines in mature neurons differ from juvenile stubby spines. There is also an assumption that, in mature neurons, the stubby spine is just a step in mushroom spine elimination.

    One more issue where this study might be extended is the differences between thin and mushrooms spines, because the morphologic boundary between them is elusive. The authors tried to investigate protein abundance relative to spine subclasses determined by neck length, head width, and PSD area (Еxt Fig. 10), but the conclusions are not clear.

    In general, these finding are very important in order to determine the relationship between shape and function in spines, an issue that we and others discussed extensively last year (Pchitskaya and Bezprozvanny, 2020). 

    This paper opens new questions in the investigation of spine processes, and opens possibilities in their modeling. Synaptic change and elimination are at the center of Alzheimer’s disease pathogenesis (Selkoe, 2002), and it will be very interesting to apply such methods to studying spines during various stages of neurodegeneration. These findings may be particularly important to the goal of finding primary processes inducing spine instability, and they open a new window into spine-preserving therapy to prevent their elimination in various conditions.

    References:

    . Dendritic Spines Shape Analysis-Classification or Clusterization? Perspective. Front Synaptic Neurosci. 2020;12:31. Epub 2020 Sep 30 PubMed.

    . Alzheimer's disease is a synaptic failure. Science. 2002 Oct 25;298(5594):789-91. PubMed.

    View all comments by Ilya Bezprozvanny
  4. The work presented here used a combination of cutting-edge technologies, including super-resolution imaging and quantitative biochemistry. It generated quantitative molecular-scale images of two types of spines, mushroom and stubby, from cultured rat neurons.

    The finding that the two types of spines have similar protein compositions is very interesting, although a more significant correlation of trafficking proteins to synaptic strength in mushroom spines may not be surprising. It would be important to validate these findings in other culture systems, such as iPSC-derived neurons and in vivo.

    The strength of the paper lies in the technology innovation. The system can be utilized to address some of the key questions relevant to Alzheimer’s disease. In particular, It would be interesting to assess how the copy number and distribution of the dendritic spine proteins, especially those related to protein trafficking, exocytosis and endocytosis, change in response to extracellular (Aβ) or intracellular (tau) protein aggregation. This will reveal additional mechanistic insight into synaptic dysfunction in AD.

    View all comments by Hui Zheng

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  1. Peering Inside Stubby and Mushroom Dendritic Spines