The highly arborised processes of astrocytes, the major glial cell population in the CNS, play a vital role in the surveillance and modulation of synapse function. The efficacy of this role depends on the availability of astrocyte processes in the vicinity of pre- and post-synaptic terminals. However, little is known about the regulation of process structure and the molecular machinery underlying their function. Understanding these particular mechanisms is highly pertinent to exploring what goes wrong between astrocytes and neurons in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), in which synapse dysfunction is an early feature. Amid growing evidence of toxic gain of astrocytic function1–4, it is also pertinent to highlight studies describing the loss of neurosupportive function, which may help refine experimental targeting approaches5–7.
In our recently published paper in Communications Biology7, “A human proteogenomic-cellular framework identifies KIF5A as a modulator of astrocyte process integrity with relevance to ALS”, we reported a novel loss of function pathology relevant to ALS. Specifically, we generated a systems biology framework allowing the discovery of common astrocyte-specific disturbances in ALS. Our approach helped reveal the presence and role of astrocytic KIF5A, motor protein kinesin-1 heavy-chain isoform 5A, previously described only in neurons. Here, we summarise our significant findings and how we arrived at our conclusions.
A proteogenomic pipeline advances mechanistic discoveries in astrocyte pathobiology
We designed a workflow based on a genome-wide association study (GWAS)-linked network propagation method. Our initial results predicted a network of genes that could interact in ALS, for instance, at transcriptional and/or protein interaction level, leading to shared downstream effects.
Figure 1
We found overlapping gene network modules across ALS cohorts, which converged on a set of genes associated with a microtubule organisation and cell transport function (1a). Some of these modules and pathways were also shared amongst other neurodegenerative diseases.
The integration of these modules with ALS astrocyte-specific transcriptomic and proteomic data indicated the failure of cytoplasmic trafficking machinery in astrocytes (1b,c).
In particular, the expression and protein levels of KIF5A, the subunit of kinesin-1, were diminished in ALS astrocytes compared to controls (1c).
KIF5A is present in astrocytes and regulates its process structure and function
While in our recent study6, we detected KIF5A protein levels in astrocyte cultures by highly sensitive mass spectrometry, cellular visualisation of KIF5A had been challenging in studies using human postmortem CNS samples, and as such, it could not be confidently identified in astrocytes. To overcome these limitations, we generated a highly accessible mouse and also human patient-specific induced pluripotent stem cell (iPSC)-derived cell culture platform and applied perturbation experiments followed by super-resolution microscopy, which allowed us to confirm both the distribution and role of KIF5A in astrocytes.
Figure 2
We found that KIF5A is widely distributed along astrocyte processes, albeit in low levels, and its forced reduction can lead to disruption of structural integrity and cell polarity (2a,b), which could theoretically diminish process availability at synapses. Our findings also corroborate recent exciting data on the role of kinesin-1 in microtubule dynamics8.
We have shown that reduced KIF5A levels impair the transport of mitochondria, a cargo of kinesin-1/KIF5A (2b), suggesting a potential blockade of mitochondrial energy supply at distal process segments involved in synaptic maintenance.
SOD1 ALS astrocytes recapitulate the KIF5A-deficiency phenotype, and it is rescuable
Our discoveries on the astrocytic role of KIF5A prompted us to investigate whether KIF5A deficiency observed in SOD1 ALS astrocytes could be a factor in their phenotypic alterations, including structural and functional disturbances of processes.
Figure 3
Super-resolution microscopy revealed similar disturbances in SOD1 ALS astrocytes as seen for control cells in which KIF5A expression was knocked down, including process shortening, limited KIF5A distribution and potentially impaired mitochondrial association with glutamate transporter, EAAT2 at the astrocytic process tip (3a-c). The latter finding may pose a synergistic functional deficit9 to the loss of EAAT2 gene expression, one of the first astrocytic pathologies observed in ALS5.
We then demonstrated that the limited KIF5A distribution in distal process segments is one of the likely causes of phenotypic alterations observed in ALS astrocytes. This conclusion was supported by the improved process arborisation and spread of KIF5A and mitochondria in response to the overexpression of Jun N-terminal kinase-1 (JNK1), a kinesin regulator10 (3c). Since kinases are highly targetable, strategies that could activate kinesin-1 motility in ALS astrocytes should be further examined as a potential approach in experimental ALS therapies.
Conclusions
Here, we developed a framework consisting of a proteogenomic pipeline that allows broader predictions of pathway disturbances in ALS, which can then be integrated with a human cell-based experimental platform for cell type-specific validations. These approaches could be beneficial for teasing apart adaptive and maladaptive loss or gain of function changes during astrocyte responses in pathology, which have been hampering therapeutic advances11. Now, three-dimensional human models, such as neural organoids that mimic bona fide cell interactions, provide a unique opportunity to examine the therapeutically relevant impact of astrocyte pathologies on neuronal networks12.
References
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