Alice Y.Liu

“Age-related neurodegenerative disease” underscores human age as the primary risk factor for disease development: Alzheimer’s,Parkinson’s,and Huntington’s disease (HD) as examples.Reasons for the age-dependent delay in disease manifestation,in particular for autosomal dominant forms of the disease,and the underlying cause(s) of specific neuron dysfunction and death to manifest as memory loss,anxiety,depression,and agitation in disease subjects remain unclear.We are interested in understanding age-related changes in cell environment that can modulate the structure,function,aggregation,and pathogenicity of disease proteins implicated in neurodegenerative disease (ND),using the polyQexpanded mutant Huntingtin protein (mHtt) that causes HD as a model for our studies.Our perspective is that age-related changes in cell milieu including a reduction in intracellular water content,increased crowding,and decreased macromolecular hydration can augment the innate propensities of disease proteins to structure,to engage in heterotypic binding to and quenching of key regulatory protein factors,to wreak havoc in cell signaling and regulation that drive disease pathogenesis,as well as to form disease protein aggregates through homotypic self-association.We are hopeful that a better understanding of age-related changes in cell milieu that are conducive to ND pathogenesis will contribute to efforts to delay or prevent neuron degeneration while supporting neuron survival and function for healthy aging.THEME: aging → decreased cell hydration and increased crowding → intrinsically disordered protein (IDP) structuring &aggregation → ND progression.

A common feature of ND is the presence of disease protein aggregates,such as amyloid plaque of Alzheimer’s disease and Lewy body of Parkinson’s disease,in the central nervous system of disease subjects;an observation made well before the underlying molecular,genetic,biochemical,or cellular processes are understood.Guided by the sequence-structure-function paradigm of proteins-that the primary structure of a protein dictates the three-dimensional folded structure,and structure determines function-a longheld supposition has been that disease proteins implicated in ND,contrary to ubiquitously folded and structured proteins,are prone to “misfold” to drive aggregation,dysfunction,and disease pathogenesis.Indeed,this concept underpins the many expansive therapeutics development efforts over the decades of using small molecules and humanized monoclonal antibodies aimed at lowering the level of disease protein aggregates,with limited progress to show for thus far.The recent recognition that a significant fraction of our functional proteome is structurally disordered,either in the entirety or in parts,calls for a re-visit of the sequence-structure-function relationship (Babu,2016).IDP along with protein with intrinsically disordered regions is estimated to constitute some 50% of our proteome.These are proteins without a fixed or ordered threedimensional structure in their NATIVE functional states;indeed,the flexible and dynamic structures of IDPs enable their transient yet specific binding to multiple partners for function.IDPs have fundamentally important roles in key regulatory processes under normal physiological condition,and their dysfunction is broadly implicated in various disease states-notably cancer and NDprompting the proposition of the “disorder in disorders” or “D2” concept (Uversky et al.,2008).

We are interested in understanding agerelated changes in cell environment that drive disease protein aggregation in regard to disease pathogenicity.Using an inducible cell model of HD for our work,we show that agents and conditions that promote structuring of proteins in cells-such as the induction of heat shock protein chaperones,the addition of organic osmolytes,and a transient lowering of cell incubation temperature-rapidly and effectively promote the compaction and aggregation of the intrinsically disordered polyQexpanded mutant HttExon1-EGFP protein into forming round,micron size aggregates termed “inclusion bodies” (IB) in our cell model (Chen et al.,2018;Aravindan et al.,2020;Castro E Costa et al.,2023).Functionally,mHtt aggregation and IB formation help mitigate transcription factor (TF) dysfunction caused by lower molecular weight forms of the mHtt protein;examples include heat shock factor 1 (TF for heat shock protein chaperone induction),cAMP-response element binding protein (TF for memory consolidation),and nuclear factor kappa B (TF for immune cell response and survival) (Chen et al.,2018).Mechanistically,we show that mHtt IB formation involves the sequential steps of structuring (i.e.,a reduction in entropy and compaction of the disordered mHtt protein) followed by enthalpydriven H-bond mediated cross-linking of mHtt and release of bound water to the bulk (Castro E Costa et al.,2023).Collectively,these and related observations,detailed below,underscore the primacy of structuring and rigidification for H-bond-mediated cross-linking in driving mHtt aggregate formation in living cells.As such,there is good evidence for Q-length-dependent structuring and compaction of the intrinsically disordered polyQ-Htt protein in cell-free systems.Firstly,CD,EPR,and NMR spectroscopies show that with each Q residue,there is an enhanced secondary structure stability of 0.03-0.05 kcal/mol (Wang et al.,2006;Bravo-Arredondo et al.,2018).Secondly,molecular dynamics simulation of synthetic polyQ peptidesin vitrodemonstrates that the average compaction increases with polyQ length (Wang et al.,2006).Thirdly,aggregation canonical free energy profiles show a favorable downhill landscape for Q40 Htt compared to an unfavorable uphill profile for Q20-30 protein (Chen and Wolynes,2017).Analyses of other disease proteins implicated in ND provided similar insights on cause-effect relationship of disease protein structuring that primes their aggregation for reduced toxicity.For example,a large-scale mutational analysis of the amyotrophic lateral sclerosis disease protein TDP-43 shows that mutations that increase hydrophobicity promote aggregation and reduce toxicity across >50,000 genotypes (Bolognesi et al.,2019).Similarly,structuring of α-synuclein promotes fibril formation (Uversky et al.,2001),whereas mutations of both IκB and prion protein cause disorder-to-order transition and aggregation for reduced toxicity (Dembinski et al.,2014).Collectively,these studies show that (1) mutations and conditions that promote structuring of disease-causing IDPs prime their aggregation,and (2) disease protein aggregate formation can divert pathogenic interaction of the disease protein with key cellular targets for reduced toxicity.

Symptomatic manifestation of ND is age-related.For HD,the age of disease onset is inversely correlated with the CAG trinucleotide expansion in Exon1 of the Htt gene,with a peak of incidence in the fourth-fifth decade of life.Owing to its autosomal dominant inheritance,typical onset beyond childbearing age,progressive course,and a combination of motor,cognitive and behavioral features,HD is most devastating to patients and their families.The reason(s) for the age-related and delayed onset of this autosomal dominant disease trait is not well understood.A blunting of the protein turnover machinery with age and thus the increased accumulation of disease proteins with toxicity,particularly in post-mitotic neurons,are likely contributing factors.More importantly,given the disordered nature of the polyQ-expanded mHttExon1and the well-known exquisite sensitivity of IDPs to minute changes in their environment,we hereby suggest that ageassociated changes of decreased cell hydration and increased macromolecular crowding can enhance structuring of the intrinsically disordered polyQ-mHtt to engage in heterotypic binding to and quenching of key regulatory protein factors to drive disease pathogenesis,as well as to aggregate forming IBs through homotypic self-association.

Indeed,there is a body of scientific and lay evidence of an age-related decrease in intracellular hydration status in cell and animal models as well as in human subjects.Using circulating erythrocytes (aka: red blood cell [RBC];the lifetime of～120 days in circulation) as a model,it is shown that erythrocyte aging is associated with statistically significant increases in hemoglobin concentration and cell density accompanied by decreased cell volume (Minton,2020).Quantitative modeling of this age-related increase in macromolecular crowding from water loss in the aging RBC suggests that these changes may result in: (1) a general age-dependent loss of intracellular protein solubility,(2) a delayed and rapid appearance of high molecular weight aggregates,and (3) an age-dependent transfer of intracellular protein from dilute to concentrated or condensed phases.In a cross-sectional study of the fluid volume balance of Japanese adults between 15-88 years old,statistics reveal a highly significant and specific decrease in the intracellular water content and reduced cell volume with age (Ohashi et al.,2017).The cause(s) for this agerelated decrease in cell/tissue hydration is likely due in part to a dampening of thirst sensation and reduced fluid intake,as well as to an ageassociated decrease in lean muscle mass (that holds water) and increase in fatty tissue.The relevance of this body of information in the context of our understanding of age-related ND is underscored in the title of a published opinion,that “Supersaturation is a major driving force for protein aggregation in neurodegenerative diseases” (Ciryam et al.,2015).Further,disease protein aggregation can have both protective and detrimental effects,depending on the specific context and disease state.

Proteins that constitute our proteome span the entire structural spectrum,from the stably folded to the unfolded and intrinsically disordered;folded proteins relying on their compact folded structures for function versus the dynamic and open structures of IDPs for their transient,yet specific,interactions with binding partners for function.It stands to reason that maintenance of the NATIVE state of ALL proteins across the structural spectrum is necessary for optimal function and homeostasis;that deviation from their native structural states,be it natively folded or disordered,causes dysfunction and aggregation for pathogenicity.It further stands to reason that agents and conditions that can rectify disease protein conformation to emulate that of the normal counterpart could potentially blunt disease pathogenesis,restore normal function,and null pathogenic protein-protein interaction as well as disease protein aggregate formation.

The 2020 Lancet Commissions report on “Dementia prevention,intervention,and care” lists 12 modifiable risk factors that account for～40% of worldwide dementias,factors that could theoretically be prevented or delayed,with “l(fā)ess education,hypertension…” at the top of the list (Livingston et al.,2020).Statistics from the US Center for Disease Control and Prevention show the mean daily water intake of the US population scales (1) inversely with age groups: from 51 ounces/day from 20-39 years old to 36 ounces/day for individuals >60 years,and (2) proportionally with education level: from 36 ounces/day on average of individuals with less than a high school education to 48 ounces/day for >college educated individuals (https://www.cdc.gov/nutrition/data-statistics/plain-waterthe-healthier-choice.html).Furthermore,there is a strong association between hypertension status and hydration parameters.A recent crosssectional study shows that the intracellular water percentage and total body water percentage were significantly and negatively related to hypertension status (Mohammedin et al.,2022).Together,these considerations suggest that maintaining optimal hydration in old age can be a simple and achievable FIRST step for neurodegenerative disease prevention and healthy aging!

I am grateful to my many collaborators over the years,in particular Professor Kuang Yu Chen from the Department of Chemistry and Chemical Biology,Rutgers University.I am thankful for the many conversations and help in the reading and editing of this perspective by my students,in particular Rida Shah.

Alice Y.Liu*

Department of Cell Biology and Neuroscience,Rutgers State University of New Jersey,Piscataway,NJ,USA

*Correspondence to:Alice Y.Liu,PhD,liu@dls.rutgers.edu.

https://orcid.org/0000-0002-6984-1671(Alice Y.Liu)

Date of submission:April 17,2023

Date of decision:May 25,2023

Date of acceptance:June 27,2023

Date of web publication:September 4,2023

https://doi.org/10.4103/1673-5374.382234

How to cite this article:Liu AY (2024) A perspective on age-related changes in cell environment and risk of neurodegenerative diseases.Neural Regen Res 19(4):719-720.

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