A synthetic extracellular scaffolding protein (ESP) has been shown to restore synaptic functions, motor coordination, memory and locomotion in mouse models of cerebellar ataxia, Alzheimer's disease (AD), and spinal cord injury (SCI), in a collaborative European and Japanese study.
Termed CPTX, the synthetic ESP could provide an effective treatment for nerve injuries, and for neurodevelopmental and neurodegenerative conditions, the authors reported in the August 28, 2020, edition of Science.
"We believe CPTX is the first and only synthetic synaptic ESP shown to be effective both in vitro and in mouse models of ataxia, AD and SCI," said lead author Michisuke Yuzaki, a professor in the Department of Physiology at Keio University School of Medicine in Tokyo.
Yuzaki co-led the study together with Alexander Dityatev, a professor and head of Molecular Neuroplasticity at the German Center for Neurodegenerative Diseases in Magdeburg, and Radu Aricescu, a professor in the Neurobiology Division of the MRC Laboratory of Molecular Biology in Cambridge, U.K.
Synapses enable interneuronal communication and are highly dynamic molecularly, with synaptic remodeling being essential for all aspects of brain physiology.
However, remodeling errors can occur, leading to the excitatory and inhibitory signaling imbalance thought to be a major cause of neuropsychiatric or neurological disorders, including autism, epilepsy, schizophrenia and AD.
Synapse formation is driven by synaptic organizer proteins, among which the ESPs, cerebellin-1 (Cbln1) and neuronal pentraxin-1 (NP1), can rapidly induce synapse differentiation by binding pre- and/or postsynaptic cleft cell surface proteins.
Synthetic molecules combining Cbln1 and NP1 structural features might therefore reverse excitatory synapse loss and promote structural and functional recovery of damaged neuronal circuits.
NP1 recruits postsynaptic AMPA subtype glutamate (Glu) receptors (AMPARs) through its pentraxin domain, which is responsible for excitatory neurotransmission, but does not induce presynaptic specializations in vivo.
Conversely, Cbln1 promotes presynaptic differentiation by interacting with the cell adhesion molecule, neurexin (Nrx), through its N-terminal multimerization domain, but cannot bind AMPARs.
In their new Science study, the research team developed CPTX, a synthetic molecular structure-guided hexameric, soluble ESP synaptic organizer bioengineered to include the functional domains of both Cbln1 and NP1.
They hypothesized that CPTX might induce trans-synaptic molecular bridges, thereby accumulating and aligning presynaptic vesicle release mechanisms and postsynaptic neurotransmitter receptors.
Indeed, recombinant CPTX was shown to selectively bind presynaptic Nrx with nanomolar affinity, while binding with most AMPAR subtypes with micromolar affinity.
When administered to hippocampal neurons in vitro, CPTX was shown to act as a bidirectional synapse organizer, inducing excitatory pre- and postsynaptic sites.
This is a significant finding, as "a synaptic organizer that can induce connection of synapses must be able to bind both pre- and postsynaptic receptors and induce bidirectional maturation," Yuzaki told BioWorld Science.
CPTX also increased the number of functional excitatory synapses and improve gait performance upon injection into the brains of ataxic Cbln1- and glutamate delta-2 (GluD2) receptor-gene knockout mice.
Compared to wild-type (WT) controls, "synapses were reduced to 40-60% and 20-30% in GluD2- and Cbln1-null mice, respectively, while CPTX injection restored these synapses to around 70% and around 45% versus controls," noted Yuzaki.
"We have previously shown that Cbln1 is effective in restoring synapses in Cbln1- but not in GluD2-null mice, because Cbln1 requires its postsynaptic receptor GluD2.
"In contrast, CPTX induced synapses in both Cbln1- and GluD2-null mice and significantly improved the gait of both mouse models 3 days after the injection.
"In terms of speed, irregularity, stride length and coordination, all gait parameters were improved to around 30-65% of those in WT mice," Yuzaki said.
Moreover, injecting CPTX into the hippocampi of mouse models of familial AD was shown to restore dendritic spine numbers and excitatory synaptic transmission.
"Dendritic spine density in CA1 pyramidal neurons was reduced to around 70% of WT controls in AD mice, while CPTX administration into the AD hippocampi restored spine density to the level seen in WT mice," said Dityatev.
"Basal synaptic transmission was two-fold reduced in AD mice but could be increased to even exceed the normal level by CPTX, which also rescued synaptic plasticity."
Hippocampal CPTX injection was also observed to significantly improve hippocampus-dependent learning and memory.
"To evaluate spatial memory in a minimal stress paradigm, we used a labyrinth test with food pellet rewards, in which AD mice traveled longer distances than WT mice during retrieval and reversal sessions," explained Dityatev.
"CPTX injection 3 days before testing modestly improved spatial memory in AD mice, possibly due to insufficient dispersal of injected CPTX, because spatial memory requires integration of information from broadly distributed head direction, grid and place cells.
"To more directly assess the CA1 region's function, we evaluated contextual fear conditioning; AD mice failed to discriminate between a context in which they received an electrical shock and a neutral context, whereas CPTX injection completely rescued context discrimination in AD mice," said Dityatev.
Bridge over troubled waters
Importantly, in mouse SCI models, CPTX injection into the damaged tissue was shown to reorganize excitatory circuits and restore locomotion for more than 7-8 weeks.
"Essentially, there are no effective SCI treatments, especially at chronic stages," Yuzaki said.
"Unlike the few existing SCI treatments, which primarily aim to regenerate damaged axons, CPTX appears to work by promoting re-organization of spared intra-spinal networks," he added.
"Moreover, while these treatments require repeated or continuous application to the damaged spinal cord at the acute stage, a single CPTX injection 1 week after the injury significantly restored locomotion."
These findings suggest CPTX may represent a prototype for developing new molecular research tools, as well as for treating neurological disorders including AD.
"CPTX could improve brain functions by restoring damaged synapses in neurodegenerative diseases such as AD, but the primary cause of AD synapse loss is abnormal amyloid beta proteins, so CPTX could serve as an adjunctive therapy," said Yuzaki.
However, "CPTX could serve as a primary therapy for SCIs, perhaps in combination with other agents, but before entering clinical trials, its safety and efficacy must be tested in larger animals, while the most effective dose and application route also await optimization."
Meanwhile, "new ESPs continue to be discovered and, by modifying their modular structures, we plan to develop synthetic ESPs with different pre- and postsynaptic specificities, which could be used to restore or modify synaptic connectivity in neurological and neuropsychiatric disorders." (Suzuki, K. et al. Neuroscience 2020, 369(6507): 1074).