Novel adult cortical neuron processing and screening method illustrates sex- and age-dependent effects of pharmaceutical compounds

Abstract

Neurodegenerative diseases and neurotraumatic injuries are typically age-associated disorders that can reduce neuron survival, neurite outgrowth, and synaptic plasticity leading to loss of cognitive capacity, executive function, and motor control. In pursuit of reducing the loss of said neurological functions, novel compounds are sought that promote neuron viability, neuritogenesis, and/or synaptic plasticity. Current high content in vitro screenings typically use cells that are iPSC-derived, embryonic, or originate from post-natal tissues; however, most patients suffering from neurodegenerative diseases and neurotrauma are of middle-age and older. The chasm in maturity between the neurons used in drug screens and those in a target population is a barrier for translational success of in vitro results. It has been historically challenging to culture adult neurons let alone conduct screenings; therefore, age-appropriate drug screenings have previously not been plausible. We have modified Miltenyi’s protocol to increase neuronal yield, neuron purity, and neural viability at a reduced cost to expand our capacity to screen compounds directly in primary adult neurons. To our knowledge, we developed the first morphology-based screening system using adult cortical neurons and the first to incorporate age and sex as biological variables in a screen using adult cortical neurons. By using primary adult cortical neurons from mice that were 4 to 48 weeks old for screening pharmaceutical agents, we have demonstrated age- and sex-dependent effects on neuritogenesis and neuron survival in vitro. Utilizing age- and sex-appropriate in vitro models to find novel compounds increasing neuron survival and neurite outgrowth, made possible by our modified adult neuron processing method, will greatly increase the relevance of in vitro screening for finding neuroprotective compounds.

Morphological screens using aged primary adult neuronal, microglial, and astrocytic cultures to find novel neurotherapeutics

“…Increasing neurite growth and neuron survival in adults has great therapeutic potential. Neurite growth can improve neural communication after neurotrauma and coupled with improved survival, can mitigate necrosis after trauma and maturation of neurodegenerative diseases ( Sefiani et al, 2022 ). Therefore, there is a need for a screening system that recapitulates the regenerative capacity and metabolic activity of aging neurons to develop novel therapeutic strategies for age-associated neurological disorders.…”

“…The method involves the removal of debris and blood cells, followed by the separation of neurons from glia using magnetic-activated cell sorting (MACS) technology (Miltenyi Biotec, North Rhine-Westphalia, Germany) to produce a neuron enriched culture. Using this method, researchers illustrated the need for using aged neurons in screens because, even in vitro , drugs, such as RO48 and 7-epi Paclitaxel, induce obvious age-dependent effects on neurite growth and survival ( Sefiani et al, 2022 ). Therefore, screening in primary adult neurons provides distinct advantages in finding novel therapeutics for age-associated neurological disorders, hypothesized to more accurately reflect clinical response to a given drug.…”

“…From 1990 to 2010, total dementia cases almost doubled, and per capita dementia cases increased by 53%. From 1978 to 2005, the percentage of people suffering from a spinal cord injury (SCI) being of geriatric age increased by over 2 1/2-fold ( Sefiani et al, 2022 ). One example of an age-associated neurological disorder is stroke, where 75% of patients are over the age of 65 and patients over 75 years of age are more likely to be hospitalized and have higher mortality rates ( Simmons et al, 2023 ).…”

“…This dichotomy in age between human populations and animal models is likely to impede the understanding of the pathological mechanisms of most neurological disorders and the translation of their respective promising therapies considering aging itself is the single greatest risk factor for stroke, dementia, and many other neurological diseases ( Wahl et al, 2019 ). Additionally, drugs, such as methylphenidate used to treat attention deficit-hyperactivity disorder, can induce completely opposite effects depending on the patient’s age ( Sefiani et al, 2022 ). Therefore, even in the same species, drugs can have age-dependent effects.…”

New approach methodologies to address population variability and susceptibility

“…Genetic variability and environmental exposures have also been evaluated using cell lines [ 9 ], induced pluripotent stem cells (iPSCs) [ 10 , 11 ], in silico models [ 12 ], and small model organisms, e.g., Zebrafish [ 13 ], Elegans [ 14 ] and Drosophila [ 15 ]. NAMs have been applied to assess additional factors that contribute to variability and susceptibility such as sex [ 16 , 17 ], life stage [ 16 , 18 , 19 ], and comorbidities [ 20 ], including rare diseases [ 21 ]. Moreover, NAMs have the potential to incorporate complex mixtures and cumulative exposures [ 22 – 24 ].…”