Massive transcriptome sequencing of human spinal cord tissues provides new insights into motor neuron degeneration in ALS

Overall Aim: Further the goal of identifying molecular determinants underlying the onset and progression ALS. 

•  The authors conducted whole transcriptome sequencing from ALS donor tissue. 

• They identified 1160 deregulated genes

  • • Down regulated genes tend to have neuronal origin

    • Upregulated genes tend to originate from glial cells and are involved in neuroinflammation or cell death

• SNAP25 and STX1B showed strong mRNA and protein level deregulation 

  • • Results suggest that impaired synaptic function caused by the loss of SNAP25 could potentially lead to the calcium elevation and glutamate excitotoxicity characteristic in ALS. 

Figure 1 shows clear seperation between diseased and normal groups, as by be observed by the hierarchical clustering

Figure 2: Authors found statistically significant changes in cell populations composed by neurons, motorneurons, oligodendrocytes and microglia, but not in the astrocyte population.

• While neuron, motorneuron and oligodendrocyte populations appeared depleted in ALS samples, microglial population was over-represented.

  • • Microglial cells play an important role in driving inflammatory immune response

    • Over expression of microglia suggests neuroinflammation, which is thought to be critically important in the neurodegenerative process

      • • might have initial benefit, but contributes to disease progression if chronic

• Authors found through sequencing:

  • • 18 dysregulated miRNAs involved in TGF-beta signaling, axon survival and outgrowth.

    • RNA-seq revealed altered expression of 1142 genes 

      • • • • these genes belong to range of ALS deregulated molecular pathways including impaired synaptic function

            • downregulated genes typically involved in nervous system processes such as impulse transmission, synapse function, or calcium metabolism.

            • Authors identified 15 synaptic proteins related to the synaptic vesicle cycle.

              • • Consist of docking and fusion of the vesicles, and subsequent exocytosis and neurotransmission.

                •  Changes in synaptic transmission have been investigated in Alzheimer’s disease and may have a primary role in ALS for which little is known. 

• Authors particularly focused on two observed downregulated neuronal t-SNARE proteins: SNAP25 and Syntaxin-1B (STX1B)

  • • form a ternary complex with the v-SNARE protein synaptobrevin

    • comlex mediates the neurotransmitter release at the presynaptic membrane.

    • SNAP25 negatively regulates native voltage-gated calcium channels (VGCCs) in glutamatergic neurons

      • • • its reduced expression enhances glutamatergic neurotransmission and causes neurodegeneration. 

            • • • Overstimulation of this type of glutamate receptor would then lead to neuronal calcium overload

                • Excessive accumulation of intracellular calcium is the key observed process leading to neuronal death or injury

    • also observed upregulation of STX1A (homologous to STX1B)

Fun fact: Botox works by cleaving these snare proteins. Through their proteolytic action on these proteins, botulinum toxins prevent the fusion of secretory vesicles with the plasma membrane (exocytosis), thereby inhibiting neurotransmitter release. 

Observed downregulation of SNAP25 and STX1B and upregulation of STX1A in comparison to control

Discussion

• Data show a depletion of neuron, motorneuron and oligodendrocyte populations with down regulated genes of neuronal derivation and linked to relevant neurological functions as impulse transmission, synaptic transmission, calcium ion transport or neurotransmitter secretion. Up regulated genes, instead, are generally involved in neuroinflammation, immune responses or cell death and tend to have microglial origin.

• As a consequence of neurological injury caused by a gene disregulated, surrounding cells are activated leading to a cascade of inflammatory events.

• RNA-Seq data support the down-regulation in ALS donors of SNAP25 and STX1B

  • • These are neuronal t-SNAREs involved in vesicle trafficking and calcium dynamics

    • A redundant role between STX1B and its homologous STX1A has been reported . While STX1B protein levels are shown to be strongly downregulated in ALS, STX1A was upregulated in five out of six tested ALS patients

      • • STX1A up regulation may be compensating for some of the STX1B loss

        • STX1A may also compensate for SNAP25 reduction in order to restore calcium homeostasis.

• Experiments have shown that loss of SNAP25 impairs calcium-evoked exocytosis. Deletion of STX1B impairs both spontaneous and evoked fast synaptic vesicle exocytosis

  • • Deletion of STX1A has no effect on both types of synaptic transmission probably due to compensation by STX1B. 

• Notably, the observed SNAP25 reduction highlights a potentially novel and relevant role of t-SNARE pattern in elevating intracellular calcium concentration and in triggering the glutamate excitotoxicity. 

  • • SNAP25 loss in glutamatergic neurons has shown to increase the concentration of intracellular calcium evoked by depolarization

• An initial accident altering the t-SNARE pattern causing SNAP25 reduction should lead to the increased intracellular calcium levels found in ALS motorneurons.

  • •  Subsequently, elevated intracellular calcium concentration causes motor neuron injury leading to increased glutamate release and excitotoxicity. 

• • Depending on whether your gene of interest is upregulated or down regulated can have big implications for its role in ALS. This paper describes that upregulated genes, particularly in microglia, often tend to be associated with an inflammatory response that arises as a result of cell damage and death.

    • Another example of this can be seen with STX1A. While it is observed that STX1A is upregulated in ALS, it is very likely as a compensatory measure to the loss of STX1B, which leads to neurodegeneration. This interplay can only be understood once looking further into the interactions and roles of particular genes. 

  • for us, SOAT1 is upregulated and it is microglial. This suggests it is probably overexpressed as a response to neuronal damage. 

    • This is relevant because it changes the effects we might expect SOAT1 to have if altered in ALS

Other Paper Findings

• Authors oberserved  dysregulation of cholesterol synthesis, which has been linked to chronic neurodegenerative disorders 

  • • This alteration could contribute to the impaired axon guidance and synaptic transmission observed in ALS, as cholesterol is essential for the organization and physiological fluidity of cellular membranes.

• Experiments show important roles for miRNAs regulating signaling pathways determining cell survival and axon guidance during ALS progression. 

  • TGF-beta signaling heavily implicated in motor neuron diseases. 

    • It participates in the interplay between neuronal and glial cells during ALS progression and increased levels of TGF-beta provoke an acute improvement in the motor performance of SOD1 mice 

    • Activation of the TGFβ/Smad signaling system is protective against aggregate formation of cytoplasmically mislocalized TDP-43