Neurosteroids as regenerative agents in the brain: therapeutic implications

Abstract

Regenerative therapeutics hold the promise of self-renewal and repair. Ageing and age-associated neurodegenerative diseases are marked by a decline in self-renewal and repair, but a capacity for regeneration is retained. The challenge faced by researchers developing molecular therapeutics to promote self-renewal in the nervous system is to activate regenerative and repair pathways often in the context of progressive degeneration. Neurosteroids regulate both regeneration and repair systems in the brain, and among this class of molecules, allopregnanolone has been broadly investigated for its role to promote regeneration in both the central and peripheral nervous systems. In the brain, allopregnanolone induced generation and survival of new neurons in the hippocampus of both aged mice and mice with Alzheimer disease, accompanied by restoration of associative learning and memory function. In the brain of mice with Alzheimer disease, allopregnanolone increased liver X receptor and pregnane X receptor expression, reduced amyloid-β and microglial activation, and increased markers of myelin and white matter generation. Therapeutic windows for efficacy of allopregnanolone were evident in the brains of mice with both normal ageing and Alzheimer disease. Allopregnanolone dose and a regenerative treatment regimen of intermittent allopregnanolone exposure were determining factors regulating therapeutic efficacy. Allopregnanolone serves as proof of concept for therapeutics that target endogenous regeneration, windows of therapeutic opportunity for regeneration, and critical system biology factors that will determine the efficacy of regeneration.

Conflict of interest statement

Competing interests

The author declares that she has filed applications for patents on therapeutic regimen and method of use of allopregnanolone for neurodegenerative diseases.

Figures

Figure 1 |

Mechanism of allopregnanolone-induced neural stem cell and oligodendrocyte precursor progenitor mitosis. Allopregnanolone (APα) binds to sites within the transmembrane domains of the GABAA receptor complex to both potentiate and directly activate the GABAA receptor complex (1). APα potentiates GABAA receptor complex responses through binding in a cavity formed by the α-subunit transmembrane domains, whereas direct receptor activation occurs through binding at the interface between the α and β subunits and is enhanced by APα binding to the potentiation site. Owing to expression of the SLC12A2 cotransporter in neural stem cells, intracellular Cl− is elevated relative to extracellular Cl−, such that activation of the GABAa receptor complex leads to an efflux of Ch. Efflux of Cl− from the intracellular compartment leads to membrane depolarization (2). Efflux of negatively charged ions leads to membrane depolarization and activation of voltage-dependent L-type calcium (Ca2+) channels β). The subsequent rise in intracellular Ca2+ activates a Ca2+-dependent kinase, CaMK IV, which then phosphorylates and activates the transcription factor cyclic AMP-responsive element-binding protein 1 (CREB1). Through CREB1 activation, APα upregulates expression of cell cycle genes required for transition from GO to S and M phases of the cell cycle (4). Simultaneously, genes that express proteins that repress cell division, such as the cyclin-dependent kinase inhibitors P16 and P18 and ubiquitins, are downregulated. Successful transition through the cell cycle leads to neural stem cell proliferation in the subgranular zone of the dentate gyrus and oligodendrocyte precursor progenitors in white matter (5). The mechanism of APα-induced neurogenesis takes advantage of the developmentally regulated Cl− gradient to activate a Ca2+-to-CREB signalling cascade to induce mitosis in those cells phenotypically competent to divide whilst not activating this pathway in mature neurons., Abbreviations: Ca2+, calcium; CDK, cyclin-dependent kinase; Cl−, chloride; Cyc, cyclin; Ub, ubiquitin.

Figure 2 |

Paradigm of allopregnanolone treatment, behavioural assessments and survival of newly generated neurons in wild-type mice and in the 3×TgAD mice across the life course. APα-induced neurogenesis and the relationship with associative learning and memory was tested using a behavioural paradigm aligned with the time course of proliferation, migration and integration. Data summary refers to age-matched mice of 3, 6 and 9 months of age. a | At each age tested, 3×TgAD and wild-type mice received a single injection of APα followed by BrdU on day 1 and returned to their cage for 1 week. Following the associative learning phase, mice were returned to their home cage for a week (days 13–21) followed by a memory trial at day 22 post APα injection.,b | During the week following APα treatment, newly generated cells would have migrated from the subgranular zone into the granule cell layer of the dentate gyrus. Associative learning assessed using the classic trace eye-blink conditioning paradigm was selected because of its hippocampal dependency and well-characterized relationship to neurogenesis. To ensure that the learning paradigm did not generate new neurons, association pairings were reduced to one-third of what is required to generate new neurons or to effect their survival. During the associative learning phase (days 8–12), cells generated 1 week earlier would have extended dendritic growth into the granule cell layer and molecular layer with an initial emergence of axons into the hilar region of CA3, and would have received GABAergic synaptic input. Also during this time, these cells show increased synaptic plasticity and long-term potentiation.c | Summary of learning performance over the days 8–12 of associative conditioning. Vehicle-treated 3×TgAD mice had modest-to-no associative learning. By contrast, learning performance of APα-treated 3×TgAD mice was comparable to that of normal age-matched wild-type mice, which was sustained during the 5 days of training., APα restored memory function to normal whereas vehicle-treated animals had no improvement in memory. Survival of BrdU-labelled cells generated on day 1 was maximal in the wild-type mice, ~75% of normal value in the 3×TgAD mice and no difference for the vehicle-treated mice. Memory performance and number of surviving BrdU-positive cells were significantly correlated (0.8, P<0.01) in the APα-treated 3×TgAD mice., Abbreviations: APα, allopregnanolone; BrdU, 5-bromo-2-deoxyuridine; GABA, γ-aminobutyric acid; Tg, transgenic; 3×TgAD, triple transgenic model of Alzheimer disease.

Figure 3 |

Targeting regeneration and disease mechanisms promotes therapeutic efficacy. Ageing and neurodegenerative diseases such as Alzheimer disease are systems-level changes in brain. As such, therapeutic interventions that regulate systems of responses rather than single targets with single outcomes might be required to achieve a therapeutic benefit. Allopregnanolone functions as a systems-level, pleiotropic therapeutic agent to promote the regenerative system of the brain to renew neuronal and myelinating oligodendrocyte populations, while also activating mechanisms that decrease the burden of pathology. Pleiotropic therapeutic agents that target the endogenous regenerative system of the brain, whilst also activating pathways that reduce neurodegenerative pathology, have the potential to substantially increase the probability of translational success to prevent, delay and treat neurodegenerative disease in the ageing brain. Abbreviations: Aβ, amyloid-β; LXR, liver X receptor; PXR, pregnane X receptor.

Figure 4 |

Optimization of allopregnanolone treatment regimens for regeneration and repair. The processes of renewal and repair require time and occur in stages in an obligatory sequence. Neurogenesis occurs over the course of hours, in the case of transitioning through the cell cycle, to months in the case of full integration into hippocampal circuitry and networks. Activating both regenerative and repair systems to maximize therapeutic benefit in the ageing or degenerating brain requires simultaneous assessment of both processes and investigation of multiple dosing and regimes to arrive at an optimal course of therapy. In the case of APα, a once per week treatment regimen with a single dose of APα (10 mg/kg) was optimal to promote neurogenesis while also activating systems that reduced Alzheimer disease pathology. By contrast, constant infusion treatment regimens over the course of months are antiregenerative and result in adverse outcomes. Abbreviation: APα, allopregnanolone.

Figure 5 |

Theoretical window of opportunity for therapeutic efficacy to promote endogenous regeneration of neural stem cells versus stem-cell-based therapies. Age-related neurodegenerative progression to subjective memory complaints, to cognitive impairment, to mild cognitive impairment, to probable Alzheimer disease and ultimately to Alzheimer disease is characterized by sequential development of detectable pathology and clinical symptoms. The coloured lines indicate clinical analyses performed at each stage of disease progression: CSF and PET analysis for amyloid-β (red line), FDG-PET/MRI for synaptic dysfunction (purple line), CSF analysis for Tau-mediated neuronal injury (pink line), volumetric MRI for brain structure (blue line), cognition (orange line) and clinical function (green line) analyses. The proposed window of opportunity for promoters of endogenous regeneration is based on preclinical data. Extrapolated to ageing human brain, the window of therapeutic opportunity would be bracketed starting from the onset of age-associated neural stem cell quiescence, coinciding with a decline in cognitive function and extending into the mild cognitive impairment range, and closing with the diminished pool of responsive neural stem cells coinciding with probable Alzheimer disease. Whereas regenerative transitions are fairly well defined in the rodent brain, the transition in the human brain and markers of neurogenesis continue to be at the leading edge of clinical neuroscience.,, Red lines in inset figure indicate projections of cognitive function relative to chronological age. Abbreviations: CSF, cerebrospinal fluid; FDG-PET, 18F-fluoro-2-deoxyglucose PET; MCI, mild cognitive impairment.