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\begin{document}

\title{Novel therapeutic approaches to target neurodegeneration}



\author[1]{Alerie Guzman de la Fuente}%
\author[2]{Silvia Pelucchi}%
\author[2]{Jerome Mertens}%
\author[3]{Mónica Di Luca}%
\author[4]{Daniela Mauceri}%
\author[3]{Elena Marcello}%
\affil[1]{Instituto de Investigación Sanitaria y Biomédica de Alicante}%
\affil[2]{Leopold Franzens Universität für Innsbruck}%
\affil[3]{Università degli Studi di Milano}%
\affil[4]{University of Marburg}%


\vspace{-1em}



  
  \date{May 19, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Ageing is the main risk factor common to most primary neurodegenerative
disorders. Indeed, age-related brain alterations have been long
considered to predispose to neurodegeneration. Although protein
misfolding and the accumulation of toxic protein aggregates have been
contemplated as causative events in neurodegeneration, several
biological pathways affected by brain ageing are also contributing to
pathogenesis. Here, we discuss the evidence showing the involvement of
the mechanisms controlling neuronal structure, gene expression,
autophagy, cell metabolism, and neuroinflammation in the onset and
progression of neurodegenerative disorders. Furthermore, we review the
therapeutic strategies currently under development or as future
approaches designed to normalize these pathways, which may then boost
brain resilience to cope with toxic protein species. Therefore, in
addition to therapies targeting the insoluble protein aggregates
specifically associated with each neurodegenerative disorder, these
novel pharmacological approaches may be part of combined therapies
designed to rescue brain function.%
\end{abstract}%



\sloppy


\textbf{Novel therapeutic approaches to target neurodegeneration}Running
title: Tackling pathways contributing to neurodegeneration Alerie G. de
la Fuente\textsuperscript{1,2,3, *}, Silvia
Pelucchi\textsuperscript{4,5, *}, Jerome Mertens\textsuperscript{5},
Monica Di Luca\textsuperscript{4}, Daniela
Mauceri\textsuperscript{6,7,\#}, Elena Marcello\textsuperscript{4,\#1}
Instituto de Investigaci\selectlanguage{ngerman}ón Sanitaria y Biomédica de Alicante (ISABIAL),
Avenida Pintor Baeza 12, 03010 Alicante, Spain\textsuperscript{2}
Instituto de Neurociencias CSIC-UMH, Avenida Ramón y Cajal, 03550, San
Juan de Alicante, Spain\textsuperscript{3} Wellcome-Wolfson Institute
for Experimental Medicine, Queen's University Belfast, 97 Lisburn Road,
BT9 7BL, Belfast, UK\textsuperscript{4} Department of Pharmacological
and Biomolecular Sciences, University of Milan, via Balzaretti 9, 20133
Milan, Italy.\textsuperscript{5} Neural Aging Laboratory, Institute of
Molecular Biology, Leopold-Franzens-University; Innsbruck,
Austria\textsuperscript{6} Institute of Anatomy and Cell Biology, Dept.
Molecular and Cellular Neuroscience, University of Marburg,
Robert-Koch-Str. 8, 35032 Marburg, Germany.\textsuperscript{7}
Department of Neurobiology, Interdisciplinary Centre for Neurosciences
(IZN), Heidelberg University, INF 366, 69120 Heidelberg, Germany. *
These authors are the co-first authors \# These authors are the
co-senior authors Corresponding author: Elena Marcello Dept.
Pharmacological and Biomolecular Sciences - University of Milan Via
Balzaretti 9 -- 20133 Milan Email: elena.marcello@unimi.it Word count:
9524\textbf{Acknowledgements}The authors would like to apologize to
colleagues whose work has not been mentioned or discussed here due to
constraints of space and scope. We have attempted to provide a balanced
representation of different topics and mechanistic aspects but also
acknowledge that this work will not be comprehensive and will not
realistically cover the vast body of pertaining literature. We
acknowledge funding from the Italian Ministry of University and Research
(PRIN 202039WMFP to E.M. and 20202THZAW to M.D.L.), from Fondazione
Cariplo (Grant no. 2018-0511 to E.M.), from Alzheimer Forschung
Initiative (Grant no. 21019) and the Chica and Heinz Schaller Foundation
to D.M., from Spanish Health Institute Carlos III (CP21/0032 to A.G.F.)
and Spanish Science State Agency (PID2021-124465OA-I00 to A.G.F.), from
the BrightFocus Foundation (grant A2019562S to J.M.); from the European
Union (grants ERC-STG-2019-852086 and H2020-MSCA-IF-2017-797205 to
J.M.); from the Austrian Science Fund (grant FWF-I5057 to J.M.), Clene
Nanomedicine to J.M., the U.S. National Academy of Medicine (NAM) to
J.M., the Michael J. Fox Foundation (MJFF) to J.M., and the NIH National
Institute on Aging (grant AG056306 to JM). Figure was created with
BioRender.com.\textbf{Conflict of interest}All the authors have no
conflict of interest.\textbf{Abstract}Ageing is the main risk factor
common to most primary neurodegenerative disorders. Indeed, age-related
brain alterations have been long considered to predispose to
neurodegeneration. Although protein misfolding and the accumulation of
toxic protein aggregates have been contemplated as causative events in
neurodegeneration, several biological pathways affected by brain ageing
are also contributing to pathogenesis. Here, we discuss the evidence
showing the involvement of the mechanisms controlling neuronal
structure, gene expression, autophagy, cell metabolism, and
neuroinflammation in the onset and progression of neurodegenerative
disorders. Furthermore, we review the therapeutic strategies currently
under development or as future approaches designed to normalize these
pathways, which may then boost brain resilience to cope with toxic
protein species. Therefore, in addition to therapies targeting the
insoluble protein aggregates specifically associated with each
neurodegenerative disorder, these novel pharmacological approaches may
be part of combined therapies designed to rescue brain
function.\textbf{Keywords:} neurodegenerative disorders, synapse,
dendrite, autophagy, metabolism, neuroinflammation, gene
expression.\textbf{LIST OF ABBREVIATIONS}

\begin{itemize}
\tightlist
\item
  AD: Alzheimer's disease
\item
  ADAM10: a disintegrin and metalloproteinase 10
\item
  ALS: amyotrophic lateral sclerosis
\item
  AMPK: AMP activated protein kinase
\item
  APP: amyloid-\selectlanguage{greek}β \selectlanguage{english}precursor protein
\item
  AUTEN-67: mTOR-dependent modulator autophagy enhancer-67
\item
  A\selectlanguage{greek}β: \selectlanguage{english}Amyloid-\selectlanguage{greek}β
\selectlanguage{english}\item
  CNS: central nervous system
\item
  CREB: cAMP response element-binding protein
\item
  DDQ: methylphosphonate
\item
  FDA: Food and Drug Administration
\item
  FTD: frontotemporal dementia
\item
  HAT: histone acetyltransferase
\item
  HD: Huntington Disease
\item
  HDACs: histone deacetylases
\item
  iNs: induced neurons
\item
  MCU: mitochondrial calcium uniporter
\item
  Mdivi-1: mitochondrial division inhibitor 1
\item
  Mdivi-1: mitochondrial division inhibitor 1
\item
  Mfn1: mitofusin-1
\item
  Mfn2: mitofusin 2
\item
  MS: Multiple sclerosis
\item
  mTOR: mammalian target of rapamycin
\item
  mTORC1: mTOR enzymatic complex1
\item
  NF-\selectlanguage{greek}κ\selectlanguage{english}B: nuclear factor-\selectlanguage{greek}κ\selectlanguage{english}B
\item
  NMDAR: NMDA receptors
\item
  OMM: outer mitochondrial membrane
\item
  OPA1: atrophy type 1
\item
  OPC: oligodendrocyte progenitor cells
\item
  OPTN: optineurin
\item
  OXPHOS: oxidative phosphorylation
\item
  PD: Parkinson's disease
\item
  PINK1: PTEN-induced protein kinase 1
\item
  PKC: protein kinase C
\item
  PKM: pyruvate kinase M
\item
  RNF10: RING Finger protein 10
\item
  ROS: reactive oxygen species
\item
  SASP: senescent associated secretory phenotype
\item
  SQSTM/p62: sequestrosome-1
\item
  TARDBP: (TAR)-DNA-binding protein
\end{itemize}

\textbf{Ageing and neurodegenerative diseases}

In the last century, advances in medical care and the creation of
healthier environments contributed to an increase in life expectancy .
Given that advanced age is the main risk factor for neurodegenerative
diseases , elderly population growth leads to a significant increase in
the number of patients affected by age-related primary neurodegenerative
diseases, such as Alzheimer's disease (AD) , Parkinson's disease (PD) or
amyotrophic lateral sclerosis (ALS) . Therefore, considering the current
demographic changes, primary neurodegenerative diseases will have
substantial socioeconomic implications for healthcare systems, due to
their large costs and severe impact on the quality of life of affected
individuals and caregivers, posing a critical social emergency. Hence,
addressing these large burdens for the society requires an intensified
research and novel approaches and solutions . In the last 20 years,
substantial advances in the comprehension of neurodegenerative disorder
pathogenesis have been made . Much of this progress is the result of
biochemical and histochemical characterization of proteins that
accumulate within various inclusions in the diseased brain and genetic
linkage studies identifying mutations in genes that cause
neurodegenerative diseases. The identification of specific,
disease-segregating mutations in previously unknown genes directed the
attention to proteins and pathways that are now considered crucial in
the pathogenesis of neurodegenerative diseases. For instance, certain
pathogenic mutations in the gene coding for the amyloid-\selectlanguage{greek}β \selectlanguage{english}precursor
protein (APP) cause AD, in the \selectlanguage{greek}α-\selectlanguage{english}synuclein gene are related to PD, in
huntingtin to Huntington Disease (HD), or in microtubule-associated
protein tau are associated to frontotemporal dementia (FTD) with
parkinsonism . The accumulation of species derived from these proteins
in the brain of patients often represents a histological hallmark for
each specific neurodegenerative disorder (Table 1). Despite the presence
of these inherited cases, most neurodegenerative disorders develop
sporadically in the absence of any known genetic etiology. The onset of
these sporadic forms is significantly influenced by risk factors ,
ageing being the one with the highest impact . Hence, age-associated
brain modifications are considered key contributors to the pathogenesis
of neurodegenerative disorders . However, mechanistic interface between
brain ageing and neurodegeneration have not been fully elucidated.
During the 2000s the ageing research field has grown considerably ; as
understanding how exactly ageing increases the risk to develop
neurodegenerative diseases can provide important clues for the
development of new therapeutic strategies for neurodegeneration
treatment. Even though protein misfolding and the accumulation and
formation of toxic species of proteins, due to inadequate folding, have
been seen as causative events in neurodegenerative disorders, in this
review we carefully examine the role of other, different, biological
pathways that are altered during ageing and implicated in the
pathogenesis and progression of neurodegenerative disorders. We focused
on the mechanisms controlling neuronal structure, gene expression,
autophagy system, cell metabolism, and, finally, neuroinflammation
(Figure 1). Further, we summarize the therapeutic approaches developed
to restore these pathways that may elevate the resilience of the brain
to cope with toxic species for each neurodegenerative disorder (Table
2).

\textbf{The structural disintegration: how to reshape neurons and
synapses}

The synapse is the biological locus responsible for the transmission of
information between neurons. Neuron-to-neuron synapses are composed of a
pre-synaptic and post-synaptic compartment, each with unique proteins
and structures to facilitate excitatory and inhibitory
neurotransmission. The majority of synapses are found on dendrites,
branch-like extensions of a neuron that receive information from other
neurons and carry it to the neuron soma. The excitatory postsynaptic
machinery is localized in dendritic spines, small protrusions from the
dendrite shaft. Dendrites can support information processing at multiple
spatial scales to integrate synaptic signals finally transformed into
action potentials . Neuronal synaptic structures are not static but
highly dynamic. The capability of neurons to modify the efficacy of
synaptic transmission and the synaptic structure in response to
different stimuli is called synaptic plasticity . Synaptic plasticity
has been proposed to play a central role in the brain's capacity to
incorporate transient experiences into persistent memory traces.
Astrocytes and microglia can transmit information and modulate synaptic
activity . Astrocyte processes encapsulate the synaptic cleft and ensure
recycling of released neurotransmitters, release co-factors important
for physiological neuronal transmission, and maintain tissue ion
homeostasis. Furthermore, astrocytes are connected via
gap-junction-coupled networks that synchronize neuronal activity within
brain regions . Microglia, the brain resident immune cells, on the other
hand phagocytose inactive synapses and release co-factors that are
important for the induction and maintenance of synaptic plasticity . In
addition to microglia and astrocytes, myelin, a passive insulating layer
formed by oligodendrocytes that ensures fast-saltatory conduction of
action potentials, is also essential for neuroprotection providing
physical axonal protection and trophic support. Despite being considered
for long a static component of the central nervous system (CNS), it has
now been demonstrated that myelin has a plastic nature, and that myelin
plasticity is required for motor learning and fear memory and
conditioning .

\textbf{The impact of ageing and neurodegenerative disorders on the
structure of brain cells}

Several studies documented changes in the molecular signature, the
morphology and function of brain cells with ageing. The principal
age-related neuronal structural alteration involves a reduction in
dendrites length and number, with a loss of various dendritic spines .
On the contrary, astrocytes undergo an increased expression of
cytoskeletal proteins, cell body hypertrophy, and a reduction in the
number of long, slender processes with ageing . Furthermore, aged
microglial cells show a gradual decrease in function, most notably in
chemotactic and phagocytic capacity. In particular, mouse studies have
documented impairments in the ability of ageing microglia to phagocytose
Amyloid-\selectlanguage{greek}β \selectlanguage{english}(A\selectlanguage{greek}β\selectlanguage{english}) fibrils and myelin debris In addition, recent studies
have revealed that myelin remodelling persists throughout the lifespan .
However, white matter and thus myelin volume is shown to decline after
13 months in mice, and in humans, after the age of 44-47 with myelin
alterations that contribute to age-linked functional decline being
detected prior to neuronal loss. These alterations include widespread
and diffuse myelin breakdown, degeneration, and reduced myelin renewal ,
decreased myelin stability associated with lipid peroxidation formation
of splits containing cytoplasm and myelin balloons or spheroids,
accumulation of myelin debris, like multilamellar myelin fragments . The
age-dependent altered function of glial cells reduces their ability to
homeostatically nurture, protect and regenerate neurons, generating a
more inflammatory microenvironment that consequently promotes neuron and
synapse loss and, thereby, neurodegeneration. Most of these alterations
are mild in healthy ageing, but exaggerate in a range of
neurodegenerative diseases, such as AD, PD, ALS, HD, where they
contribute or accelerate neurodegeneration, facilitate protein
aggregates deposition, impair cognition and motor function by disrupting
connective pathways Neurodegenerative diseases are characterized by
abnormalities in dendritic structure and synapse loss in different brain
regions depending on the neurodegenerative diseases . In HD, for
example, synapse loss is mainly detected in the striatal brain region,
which is linked with progressive movement discoordination . There is
growing evidence from ALS patients, FTD patients, and animal models that
suggest synaptic dysfunction and alterations in dendritic branching
begins very early in the disease before symptom onset and motor neuron
death . In AD neurons, the dendritic tree undergoes fast decline with a
decrease in the number of dendritic shafts while the few remaining show
fewer and shorter branches . Furthermore, synaptic loss in the
hippocampus and neocortex is known to be an early process in AD and the
main structural correlate with AD cognitive dysfunction. In AD and PD,
the oligomeric forms of A\selectlanguage{greek}β \selectlanguage{english}and \selectlanguage{greek}α-\selectlanguage{english}synuclein are the most toxic species
for synapses. The oligomers of \selectlanguage{greek}α-\selectlanguage{english}synuclein but not its fibrils
contribute significantly to dopaminergic loss and neuronal cell death .
Similarly, it has been shown that the A\selectlanguage{greek}β \selectlanguage{english}oligomers cause synaptic loss
and impair the mechanisms of synaptic plasticity . Interestingly, APP
and the enzymes implicated in the amyloid cascade are synaptic elements
located both, at pre and post synaptic side, and play a critical role in
regulating synaptic function . For instance, a disintegrin and
metalloproteinase 10 (ADAM10), the metalloprotease that prevents A\selectlanguage{greek}β
\selectlanguage{english}generation, is a shedding enzyme that cleaves adhesion molecules, such
as N-Cadherin, and shapes spine morphology . Furthermore, ADAM10
synaptic localization and activity are finely tuned by synaptic
plasticity phenomena and its synaptic abundance and activity towards APP
are affected in the hippocampus of AD patients . This is the result of
an impairment of ADAM10 local forward trafficking, that depends on the
PKC-regulated association with SAP97 and is also related to an increase
in ADAM10 endocytosis . Notably, alterations in ADAM10 have been
described also in HD, with increased levels of the mature form of ADAM10
in the brain areas that predominantly degenerate in HD have been
reported in mouse models of HD and in human HD brain samples .
Accumulation of active ADAM10 at the postsynaptic compartment leads to
increased proteolysis of N-Cadherin, likely promoting synaptic
instability in HD. These data confirm that synaptic failure is a common
trait of neurodegenerative disorders but highlight the importance of
investigating the molecular mechanisms underlying synaptic dysfunction
in each disorder. Therefore, given that synapses are the most vulnerable
regions of neurons, differences among synapse structure, metabolism and
signaling mechanisms might be determinants of neuronal vulnerability in
the different neurodegenerative disorders.

\textbf{Therapeutic approaches to tackle synaptic and neuronal
dysfunction}

Although we currently lack therapeutic approaches aimed at enhancing
myelination in the clinic, recent advances and high-throughput screening
approaches have provided us with potential pro-myelinating compounds.
Clemastine fumarate, a muscarinic receptor antagonist that was
identified in a pro-remyelinating drug screening, has met clinically
defined efficacy endpoints in a clinical trial in patients with Multiple
sclerosis (MS) . Recent evidence has also shown that Clemastine prevents
age-related myelin loss, neurodegeneration, and cognitive decline in
healthy ageing as well as in a mouse model of AD . Additionally, other
drugs that have been shown to promote myelin repair in the context of
MS, such as metformin or LY294002 , could also be beneficial to prevent
myelin breakdown and degeneration with age or in other neurodegenerative
diseases, but their efficacy in this context is yet to be investigated.
As neuronal connections represent the hardware for proper cognitive
abilities, therapeutic strategies aimed at preserving dendritic and
synaptic connections could conceivably be useful in neurodegenerations.
The process of neurite repair to replenish the degenerated dendrites
would involve regrowth and rewiring of the new connections within the
network. However, the local molecular and cellular milieu in the CNS
opposes neurite growth and thus renders this approach particularly
challenging . A better option could be to use strategies aimed at
upholding neuronal dendritic integrity rather than promoting its
regrowth. In this view, promising findings showed that specifically
preserving dendritic architecture in mouse models of acute
neurodegeneration (i.e. stroke) counteracted the loss of neurons which
is typically accelerated by disrupted connectivity and, ultimately,
resulted beneficial also at a functional level . Regarding synaptic
dysfunction, the 2022 drug pipeline for AD showed that synaptic
plasticity/ neuroprotective agents for which phase 2 and phase 3
clinical trials are currently ongoing are 17\% and 19\% of all
disease-modifying therapies respectively, indicating that significant
efforts are being made for targeting these mechanisms . A systematic
review analysed the efficacy profile of 12 published results of clinical
trials investigating the safety and efficacy of disease-modifying drugs
targeting synaptic plasticity in dementia . However, only three
molecules (Levetiracetam, Bryostatin 1 and Masitinib) show promising
results. Levetiracetam is a second-generation antiepileptic drug
approved as an adjunct therapy for partial seizures. The mechanism of
action seems to involve neuronal binding to the synaptic vesicle protein
2 A, inhibiting the release of calcium from intraneuronal deposits,
opposing the activity of negative modulators of GABA and
glycine-dependent currents, and inhibiting excessive synchronized
activity between neurons . An important role of this molecule also
appears to be linked to synaptic plasticity. The administration of
Levetiracetam to AD mice was reported to reverse synaptic dysfunction .
The initial clinical trials with Levetiracetam showed limited results
because of the high clinical heterogeneity of the enrolled cohort. On
the other hand, a recent clinical trial found that Levetiracetam was
able to significantly improve cognitive status only in patients with
cortical hyperexcitability. This suggests that preselection of AD
patients presenting symptoms ranging from subclinical epileptiform
activity to seizures and network hyperexcitability could improve the
capacity to identify therapeutic effects of Levetiracetam. Bryostatin 1
is a macrocyclic lactone that activates protein kinase C (PKC) and,
thereby, regulates neurogenesis, axonal transport, and synaptic
plasticity . The administration of Bryostatin 1 in AD mice restored the
number of dendritic spines in the hippocampal CA1 area . The results of
phase 1 and 2 trials were conflicting, while a recent pooled analyses
from 2 randomized clinical trials confirmed a significant cognitive
restoration elicited by Bryostatin 1 in the absence of a Memantine
treatment . Masitinib, a tyrosine kinase inhibitor, is usually used in
the treatment of mast cell tumours in animals. Studies in animal models
showed that Masitinib administration has a protective effect on synapses
because of mast cell inhibition and reduction of the secretion of
specific mediators potentially toxic to synapses . Initial studies in
patients reported that participants treated with Masitinib showed
significant improvement in cognitive function . Thus, Masitinib is being
currently investigated in a multicentre phase 3 trial for patients with
mild-to-moderate AD (NCT01872598) and showed some beneficial effects in
ALS . Pharmacological treatments of ALS patients aim at counteracting
glutamate excitotoxicity. Riluzole, an inhibitor of glutamate release,
was approved by the United States Food and Drug Administration (FDA) in
1995. Despite being associated with a short survival benefit of 2--3
months , the subsequent adoption of riluzole as a treatment for ALS was
perhaps reflective of the need for therapeutic options in the face of
this devastatingly progressive disease . Remarkably, findings from
several open-label non-randomised trials have suggested that the
greatest benefit occurs at earlier disease stages . Therefore, is
possible that riluzole's therapeutic benefit is likely to affect or
activate different cellular pathways depending on the disease stage .
Another molecule investigated to counteract excitoxicity is the
antibiotic Ceftriaxone that causes the upregulation of glutamate
transporter and decreases glutamate-induced toxicity . Phase-3 trial of
ceftriaxone indicated an overall increase in survival of patients with
ALS. Overall, these strategies tackle synaptic failure and dysfunction
as common molecular mechanism across neurodegenerative disorders. Next
challenge for drug discovery research will be to design tools and
molecules specifically targeting the mechanisms underlying synaptic
dysfunction or dendrite degeneration in each of the different
neurodegenerative disorders. An example is the control of the
trafficking of ADAM10. As mentioned above, alterations in ADAM10
synaptic localization and activity are implicated both in AD and in HD.
In AD however, ADAM10 levels are reduced in hippocampal synapses while
active ADAM10 is overexpressed in HD striatal synapses. Therefore, the
strategy to counteract synaptic failure in AD takes advantage of the
administration of a cell permeable peptide that blocks ADAM10
endocytosis, upregulates its activity, restores synaptic function
without affecting plaque deposition and rescues cognitive defects in AD
mice . On the other hand, the use of a peptide designed to inhibit
SAP97-mediated ADAM10 trafficking to the synapse , normalizes ADAM10
activity and rescues cognitive deficits in HD mice . Therefore,
understanding the mechanisms implicated in synaptic failure in the
different neurodegenerative disorders is required to design
disease-tailored therapeutic strategies.

\textbf{The main players controlling gene expression during ageing and
in neurodegenerative disorders}

A key aspect sustaining and enabling various forms of plasticity is the
regulation of gene expression. Basal transcription ensures the mere
survival of neurons as cells; to be functional computational units,
neurons further need to adapt their transcriptional responses to
physiological and pathological cues. To do so, neurons employ an array
or regulatory elements, epigenetic mechanisms, and transcription
factors, all of which are modulated by diverse stimuli. One of the most
prominent stimuli that neurons are specialized to adapt to is synaptic
activity and calcium signals. Among the many epigenetic mechanisms
employed by neurons, the ones influencing chromatin accessibility -DNA
methylation and post translational modifications of histone proteins -
received the most attention and are the best characterized. However,
more recently, non-coding RNAs have received increasing attention. Given
its key role in ensuring neural functions, it comes as no surprise that
a long list of alterations in the transcriptional landscape or
dysfunctions of molecular players have been associated with
neurodegeneration. Mechanistically, the role of transcription factors or
epigenetic regulators in the pathogenesis of neurodegenerative
conditions is explained by their capability to affect genes directly
involved in the pathology or in mediating co-morbidities. Here, we
highlight some of the better studied transcription factors and
epigenetic regulators and their involvement in neurodegeneration. cAMP
response element-binding protein (CREB) is a ubiquitous transcription
factor playing multiple roles in the CNS. Several signaling cascade
converge on CREB which acts in cooperation with cofactors such as
CBP/p300. CREB is fundamental for memory and learning but also essential
to neuronal survival and protection . Due to its broad expression, being
downstream of different signaling events and driving expression of a
myriad of critical neuronal genes, alterations of CREB activity and/or
expression CREB has been indeed reported for neurodegenerative
conditions such as AD or PD both in animal models and humans . Not all
transcription factors are always localized to the nucleus, a
considerable number of them is instead regulate their expression by
subcellular localization. Thus, failures in the nucleo-cytoplasmic
shuttling of transcription factors represent another possibility leading
to transcriptional dysfunction in neurodegenerative conditions. The
first transcription factor for which movement between the cytosol and
the nucleus was described was nuclear factor-\selectlanguage{greek}κ\selectlanguage{english}B (NF-\selectlanguage{greek}κ\selectlanguage{english}B). Activity of
NF-\selectlanguage{greek}κ\selectlanguage{english}B is prevented when sequestered in the cytosol. Alterations of NF-\selectlanguage{greek}κ\selectlanguage{english}B
localization have been observed in the proximity of plaques in
post-mortem samples from AD patients but also in dopaminergic neurons of
PD patients and are tightly linked to inflammatory states. Furthermore,
synapse to nucleus shuttling of proteins with the capacity to modulate
gene transcription is a prominent way in which inputs received at the
synapse are transferred to the nucleus to implement long-term changes.
Synapses contain several nuclear localization signals-containing cargo
proteins and different components of the nuclear import machinery, like
importin-\selectlanguage{greek}α \selectlanguage{english}and importin-\selectlanguage{greek}β, \selectlanguage{english}which have been shown to translocate to the
nucleus in an activity-dependent manner . In the last decade, few
synaptonuclear protein messengers (i.e. Abi- 1, AIDA-1D, Jacob, RNF10)
have been identified, and shown to play key roles in plasticity and
synapse function . Notably the activation of NMDA receptors (NMDAR) can
regulate gene transcription , thus affecting global protein synthesis
and, thereby memory formation. Such effect of NMDAR activation requires
the long-distance trafficking of synaptonuclear proteins. The
synaptonuclear messengers can associate with different classes of
receptors and specifically translate their activation in gene expression
changes. For instance, the RING Finger protein 10 (RNF10) operates as a
mobile hub that docks GluN2A-containing NMDAR derived signalosomes to
nuclear target sites , while protein messenger such as Jacob can encode
the synaptic and extrasynaptic origin of NMDAR signals, following
long-distance transport and nuclear import . Remarkably, alterations in
synaptonuclear messengers have been reported in neurodegenerative
disorders such as AD . From the epigenetic perspective, reduced global
methylation levels have been found in the blood of AD and PD patients in
comparison to healthy controls, probably due to the decrease in
expression of DNA methyltransferase 3a , a key mediator of this
epigenetic mark in the context of signal-regulated neuroepigenetics .
The scenario is however unclear as studies derived from post-mortem
samples of AD patients in some cases reported a reduction of global
methylation levels in cortex and hippocampus , while in other studies
lack changes or even an increase in global DNA methylation have been
observed. Studies focusing on DNA methylation changes at specific
genomic regions, functional elements, and individual gene loci have
yielded a more comprehensive view in the context of AD, but also
revealed that epigenetic changes are complex, and often contradictory
results are reported from different models (reviewed in ). Similarly,
studies have indeed reported an association between PD disease
progression and global methylation levels detected in brain or blood or
changes in the methylation pattern of specific genes . Given the
prominent role that histone deacetylases (HDACs) play in the modulation
of several neuronal functions, it comes as no surprise that they have
also been implicated in neurodegeneration. These heterogenous group of
proteins, which are classified based on their activity, structure and
co-factors, are responsible for the removal of the acetyl group from
histone as well as non-histone proteins. An example of their involvement
in neuropathologies comes from HDAC2, HDAC6 or HDAC5, whose levels are
elevated in brain areas of post-mortem AD patients . Interestingly, an
increased association between HDAC1 and CREB, possibly facilitating CREB
pathological dephosphorylation, was observed in neuronal samples of PD
patients . Expression levels of HDAC4, which has been implicated in many
forms of pathologies of the nervous systems in animal models , was shown
to be strongly associated with rapid progression of ALS in patients .
Alteration in the expression level of HDAC11 and HDAC2 were also
reported in in post-mortem brain and spinal cord tissue of ALS patients
. Beside their action as transcriptional regulators, HDACs act also on
cytosolic proteins of which the most prominent is tubulin. For example,
HDAC6 acts on tubulin and has been associated to deficits in axonal
transport in ALS patients-derived motor neurons.

\textbf{Therapeutic approaches to modify gene expression in
neurodegenerative disorders}

Targeting transcriptional regulators might be beneficial against
neurodegenerative conditions as this would result in a widespread
modulation of many affected downstream processes. However, this aspect
also represents a potential caveat as affecting the transcription of
several genes might be detrimental as not all of them necessarily
participate in the pathogenesis. Nevertheless, efforts have been made in
exploring, designing, and testing of therapeutical approaches aiming at
modulation of transcription-related processes. One of the most
sought-after targets in the treatment of neurodegenerative disorders is
CREB due to the copious evidence showing a reduction in its
functionality or expression in many diseases. At present, however, there
are no available drugs specifically boosting CREB-dependent signaling.
Multiple strategies could be followed, from acting on its upstream
signaling regulators to using molecular biology or genetic approaches to
restore its expression levels. The complexity and multifactorial nature
of many neurodegenerative disorders is likely to push the development of
drugs towards targets different than the prototypical players associated
with a certain disorder. A case for this is NF-\selectlanguage{greek}κ\selectlanguage{english}B in the treatment of
AD. Memantine, an FDA-approved molecule for the treatment of AD seems to
contrast the pro-inflammatory activity of NF-\selectlanguage{greek}κ\selectlanguage{english}B. Additional
anti-inflammatory drugs are under preclinical or clinical evaluation for
their capacity to interfere with NF-\selectlanguage{greek}κ\selectlanguage{english}B (see for review . Great efforts
have been spent in the exploration of HDACs inhibitors as useful drugs
in neurodegeneration. HDACs inhibitors may broadly facilitate gene
expression via rendering the chromatin more permissive for
transcription. In animal models, non-selective HDAC inhibitors
successfully restored memory function and neuronal structural
aberrations . However, care should be taken to follow the path of
non-selective inhibition due to its potential side effects . The design
and development of specific HDAC inhibitors has been hindered by
different problems including the fact that all HDACs share considerable
structural similarities and are expressed across different organs and
cell types. Nevertheless, with the advent of better technological
opportunities, is it quite likely that specific HDAC inhibition may be a
viable therapeutic avenue to pursue . An encouraging example comes from
inhibition of HDAC6 via Tubastatin A, ACY-1215, MPT0G211, or
5-Aroylindoles which showed promising results in AD animal models and
are currently under clinical trial evaluation for diseases other than
neurodegeneration.

\textbf{Autophagy: when the clearance system fails and drives
neurodegeneration}

Cellular homeostasis is the process that controls different cellular
activities. It is a fundamental condition that allow the cell to
maintain the physiological balance in term of cellular identity,
resilience, and survival. Defects in protein homeostasis contribute to
the lack of intracellular proteins and organelles, which progressively
accumulate in the cytoplasm. Therefore, such alterations can be
implicated in the onset and progression of neurodegenerative disorders .
Cellular component degradation within the cell can follow two different
pathways: autophagy and the ubiquitin system, specific for protein
degradation. Both processes require a concerted action of different
proteins that are recruited and specifically recognize the damaged
material, driving protein degradation as the last step to maintain
cellular homeostasis. Under stress conditions, such as ageing or
disease, this tuned process fails, resulting in an engulfment in cell
physiology. In mammalian cells there are three types of autophagy:
macroautophagy, microautophagy and chaperone-mediated autophagy.
Macroautophagy, simply known as autophagy, plays the major role in
maintaining the cellular homeostasis because it helps in the removal of
bulky protein aggregates and bigger cytoplasmic bodies. It begins with
the phagophore formation that entraps the misfolded proteins and, after
different steps, finally fuses with lysosomes to generate the
autophagolysosome . Autophagy is tightly controlled by mammalian target
of rapamycin (mTOR) and AMP activated protein kinase (AMPK). Autophagy
is promoted by AMPK, which is a key energy sensor and regulates cellular
metabolism to maintain energy homeostasis . Conversely, autophagy is
inhibited by mTOR, a central cell-growth regulator that integrates
growth factor and nutrient signals . Given its crucial role in cell
homeostasis, a defect in autophagy is associated with neuronal loss and
cognitive decline, both in physiological conditions, such as ageing, but
also in neurodegenerative disease . Interestingly, defect in the
autophagy machinery have also been linked to axonal and dendritic
degeneration and might thereby further promote the dysfunction of neural
network dysfunctions . Several neurodegenerative diseases are
characterized by defects in the degradation process of misfolded
proteins and thus, abnormal protein aggregation. Autophagy failure can
be attributed to the impairment in the clearance of pathological protein
such as \selectlanguage{greek}α-\selectlanguage{english}synuclein, A\selectlanguage{greek}β \selectlanguage{english}and tau . For instance, Beclin1 is a protein
involved in the regulation of autophagy and has been shown to be reduced
in AD patients . Furthermore, it has been shown that the downregulation
of Beclin-1 in mice resulted in reduced neuronal autophagy and A\selectlanguage{greek}β
\selectlanguage{english}accumulation . Moreover, the most common autosomal-dominant form of PD
and a familial variant that closely resembles sporadic PD is associated
to LRRK2 mutation . The altered function of mutated LRRK2 has been
linked to defects in endosomal-lysosomal trafficking and
chaperone-mediated autophagy . In dopaminergic neurons, the lysosome
number has been reported to be depleted in a mouse model of PD . In
addition to autophagy, cells take advantage of mitophagy, a specific
process responsible for the selective degradation of dysfunctional
mitochondria in PD . The PTEN-induced protein kinase 1 (PINK1),
localized on the external mitochondrial membrane, phosphorylates
mitofusin 2 (Mfn2) and ubiquitin triggering the recruitment of the
Parkin protein. This event activates several ubiquitin-binding proteins
such as optineurin (OPTN) and sequestrosome-1 (SQSTM/p62), that initiate
mitochondria to the pathway of mitophagy. Defects in the mitophagy
machinery are also a pathological pathway in PD and the accumulation of
damaged mitochondria represents one of the main pathogenetic alterations
. The PD-associated familial autosomal recessive mutations in the genes
PINK1 and Parkin have been discovered to have a key role in
mitochondrial quality control . The phosphorylation of PINK1 at the
outer mitochondrial membrane (OMM) leads to the recruitment of parkin.
Once active, parkin allows the synthesis of ubiquitin chains on OMM
proteins leading to ubiquitin chain assembly. The mutations in the PINK1
(PARK6) and parkin (PARK2) genes are linked to autosomal recessive
early-onset PD and a pathological accumulation on the OMM that triggers
an abnormal mitophagy . In PD \selectlanguage{greek}α-\selectlanguage{english}synuclein, the main component of those
pathological Lewy bodies, has been found to bind mitochondria components
, inhibiting the protein import and leading to an impaired cellular
respiration. In particular, \selectlanguage{greek}α-\selectlanguage{english}synuclein interacts with and disrupts
mitochondrial proteins such as TOM20, VDAC, and F\textsubscript{1}Fo-ATP
synthase, causing mitochondrial metabolic impairment .

\textbf{Targeting autophagy: how to promote protein and organelle
clearance}

In the last two decades, several studies have reported the
neuroprotective activity of rapamycin, one of the most powerful
pro-autophagy agents, in both cellular and animal models of
neurodegenerative diseases and, with some limitations, in human .
Rapamycin acts blocking the kinase activity of mTOR enzymatic complex1
(mTORC1), removing its autophagy suppressor activity observed under
physiological conditions. The strategy of the rapamycin treatment is to
activate the autophagic flux negatively controlled by mTORC1.
Interestingly, rapamycin has been proposed as an anti-ageing drug in
mice, since it was reported to increase the lifespan of animal models
treated . However, rapamycin treatment has been also tested in animal
models of AD. It has been shown that rapamycin administration reduced
the accumulation of A\selectlanguage{greek}β \selectlanguage{english}aggregates and prevented tau phosphorylation in
the brain of AD transgenic mice, showing a global impact in maintaining
cognition . Regarding the use of rapamycin in humans, some encouraging
results have been reported in the ALS field where a recent, randomized,
placebo-controlled, phase II clinical trial was started to evaluate the
efficacy of rapamycin in patients affected by ALS . Another approach to
modulate the mTOR signaling level is possible using mTOR-dependent
modulator autophagy enhancer-67 (AUTEN-67), a small molecule identified
as a potent candidate with anti-ageing and neuroprotective effects, by
significantly increasing autophagic flux in neurons and protecting them
from undergoing stress-induced cell death . Other agents that can
indirectly trigger AMPK-dependent mTOR inactivation are metformin and
resveratrol. Metformin activates AMPK which in turn promotes autophagy
blocking mTORC1 activity through direct inhibitions of regulatory
associated protein of mTOR. Moreover, the activation of AMPK directly
promotes the activation of phagophore-forming enzymatic complex
unc-51-like kinase (Ulk1)1/2, which is considered the initiator of the
autophagic cascade. Furthermore, PROteolysis TArgeting Chimeras (PROTAC)
technology has emerged as one of the most promising approaches to remove
specific disease-associated proteins using the autophagy machinery of
the cells. In the last few years, the PROTAC approach has been
extensively used, with several PROTACs molecules currently in clinical
trials in the cancer field . A PROTAC molecule targeting tau protein has
also been developed. It is a chimera construct made of a tau-binding
peptide, a linker, a VHL-binding peptide, and a cell-penetrating
peptide. Interestingly, this molecule leads to a significant degradation
of tau and reduced neurotoxicity of A\selectlanguage{greek}β, \selectlanguage{english}highlighting the therapeutic
potential of this approach .

\textbf{Metabolic failure and energy crisis of brain cells}

Among the different brain resident cell types, neurons are extremely
energy demanding. Neurons rely almost exclusively on the mitochondrial
oxidative phosphorylation (OXPHOS) system to fulfill their energy needs
through ATP. The OXPHOS-mediated mitochondrial functions are diverse,
ranging from the cell-intrinsic energy production to the regulation of
intracellular calcium homeostasis, synaptic plasticity, and
neurotransmitter synthesis . On the other hand, this important energy
production is accompanied by the formation of reactive oxygen species
(ROS), which in excess are detrimental for cells. ROS derive mainly from
the process of the OXPHOS, which reduces O\textsubscript{2} into
H\textsubscript{2}O using the electrons flux deriving from the
respiratory chain and leading to the formation of superoxide anion
radicals of the oxygen. Even if cell-intrinsic antioxidant defense
systems can buffer ROS, when this buffering systems is overloaded and
cell-homeostasis altered, ROS become toxic. Given the fundamental role
of mitochondria in neuronal energy supply, their dysfunction leads to an
impairment of basal neuronal energy source, impacting on several aspects
of brain physiology. In addition to their metabolic activity,
mitochondria play a key role in cellular calcium homeostasis. Cellular
calcium concentration is strictly regulated as it sustains vital aspects
of the neurons such as secretion, motility, metabolic regulation,
synaptic plasticity, proliferation, gene expression, and apoptosis .
Therefore, mitochondrial calcium dysregulation contributes to
neurodegeneration since it is the major mechanism by which increased
excitatory neurotransmission triggers mitochondrial depletion from and
retraction of dendritic structures . The function of mitochondria is
strictly related to their structure and dynamics. Thus, the
mitochondrial efficiency is measured through their capacity to undergo
continue fusion and fission cycles. Hence, mitochondrial dynamism is
important for their morphology and function, and therefore, relevant for
neuronal viability and synaptic activity. The equilibrium between
fission and fusion is key for adequate mitochondrial function and is
compromised in different neurodegenerative disorders. Drp1, the GTPase
that controls the process of fission, is altered in AD, leading to an
excessive mitochondria fragmentation and, thereby, altering their
function . This peculiar phenotype has been shown in neuronal cultures
upon A\selectlanguage{greek}β \selectlanguage{english}exposure as well as in several neurodegenerative disease animal
models . Defects in the dynamin-related GTPase proteins mitofusin-1
(Mfn1) and Mfn2, and atrophy type 1 (OPA1) protein cause mitochondrial
fusion alterations and have been reported in various neurodegenerative
disorders, including AD . Failure in mitochondria trafficking, function
and positioning in dendrites and synapses have been also observed in ALS
and FTD and may contribute to early synaptic loss in disease . In FTD,
several pathways controlling mitochondrial trafficking, dynamics, and
consequently activity are altered . In the genetic FTD caused by MAPT
mutations, a decrease in mitochondria-Tau interactions in iPSC-derived
neurons has been observed. The authors reported that neurons expressing
the Tau protein carrying the V337M mutation were characterized by
alterations in mitochondria bioenergetics affecting efficiency in
maintaining ATP levels under prolonged energetic stress. Also, Tau P301L
mutation, which is known to cause Tau hyperphosphorylation, decreases
mitochondrial respiration and ATP production, leading to a global
mitochondrial and oxidative impairment . Mutations in transactive
response (TAR)-DNA-binding protein (TARDBP), coding for the TDP-43
protein, are also associated with FTD. Interestingly, TDP-43 inclusions
aggregate outside of the nuclear compartment and can directly affect the
dynamics and trafficking of mitochondria, both at the axonal and
dendritic compartment, leading to functional impairment. The
overexpression or reduction of TDP-43, in different animal model
systems, leads to a mitochondria dysfunction In general, an altered
cellular metabolism is considered a hallmark of ageing . During cellular
senescence, which is distinct from, but associated with biological
aging, the mitochondria exhibit numerous changes in their structure,
dynamics, and function. Therefore, in senescent cells a decrease in
mitochondrial membrane potential, an increase in proton leakage and a
lowered oxidative capacity has been observed. The consequence of
modified processes resides in an altered aged metabolic homeostasis,
with a significant increase in ROS generation, diminished antioxidant
defense and a decrease in ATP production. All these phenomena have a
great impact on neurons, post-mitotic cells that are particularly
sensitive to stress and to the accumulation of a senescent profile
typical of ageing . In addition, the pool of healthy mitochondria tends
to decrease with ageing. In addition to the role played by mitochondria
in neurons themselves, the demanding energy request by neurons is also
sustained by glial cells, which are extremely flexible and respond to
environmental changes providing neurons the required energy . Every time
that neurons need energy to perform the highly energy consuming neuronal
synaptic burst, the so-called astrocyte-neuron lactate shuttle responds
to this energy demand by creating an energy bridge by which astrocyte
produced lactate is received by the neuronal part . The
astrocytic-neuronal metabolic bridge is supported by the capacity of
astrocytes to convert GABA and glutamate, removed from the synaptic
cleft, into glutamine, which is used as a precursor for refill synaptic
vesicles or for phosphorylation via the TCA cycle Neuronal metabolism
pathways are comparatively inflexible and believed to be strictly
regulated. However, metabolic changes including state shifts and
alteration is individual metabolites can have great impacts on the
neuronal epigenome. This enables the cells to adapt to environmental
changes, but also poses a risk for, as energetic challenges may lead to
highly consequential epigenetic alterations . Indeed, the cell fate
specification and the consequent cell identity are established by a
highly specific epigenetic control which must be also plastic to allow
the cell to adapt to the environment. For instance, high glucose levels
produce high acetylCoA:CoA ratio, which regulates histone
acetyltransferase (HAT) activity, and contributes to increased chromatin
accessibility and gene activation . Both the early TCA cycle
intermediate \selectlanguage{greek}α-\selectlanguage{english}ketoglutarate and oxygen are co-substrates for
demethylases, affecting DNA and histone methylation and, thereby,
changing transcription scenario . Moreover, metabolic enzymes can
translocate directly to the nucleus in a splicing- and
signaling-dependent manner, and act directly on histones triggering
changes in transcription. This is the case of pyruvate kinase M (PKM)
which translocates to the nucleus where it phosphorylates histone 3 and
leads to a de-repression of cell-cycle and glycolytic genes . In
directly converted induced neurons (iNs) from AD-patient-derived
fibroblasts, a cancer-like metabolic switch from neuronal OXPHOS to
aerobic glycolysis in AD iNs is associated with a higher level of the
PKM2 nuclear isoform compared to the physiological PKM1. PKM2 prevalence
is associated with metabolic and transcriptional changes in AD iNs,
contributing to AD-related-neuronal defects. Overall, all these new
findings are suggestive of the presence of a metabolic reprogramming
towards an aerobic glycolytic profile in AD, throughout a Warburg-effect
. The epigenetic modulation of metabolism, influenced by the combination
of pathology and ageing, could be primary or secondary to the
mitochondrial impairment . In general, in the neurodegenerative context,
there is an accumulation of macromolecular damage and metabolic
reprogramming that leads to the damage of organelles, including
mitochondria, and eventually to tissue dysfunction .

\textbf{Targeting mitochondria as therapeutic strategy for treating
neurodegeneration}

To reduce and buffer mitochondrial dysfunction the most used indirect
therapies rely on the use of antioxidants that mitigate the
mitochondrial ROS production. Some of those compounds include the
lipophilic MitoQ, CoQ10, MitoVitE, MitoTEMPOL , and resveratrol, which
indirectly activates PGC-1\selectlanguage{greek}α \selectlanguage{english}and induces mitochondrial biogenesis . There
are also compounds that modify mitochondrial dynamics, such as
mitochondrial division inhibitor 1 (Mdivi-1), and methylphosphonate
(DDQ) . In particular, Mdivi-1, an inhibitor of Dpr1, showed activity
against A\selectlanguage{greek}β-\selectlanguage{english}induced excessive mitochondrial fragmentation . A novel
approach, recently proposed, suggest combining the antioxidant effect
with epigenetic modulation. Shikonin, an anti-cancer PKM2 modulator,
would act on the metabolic shift caused by neuronal PKM2 in AD, acting
as an apoptotic brake on mature . Given the pathogenic role for
excitatory mitochondrial calcium dysregulation in mediating sublethal
dendritic atrophy observed in chronic neurodegenerative diseases,
inhibiting calcium uptake has been reported to be neuroprotective. The
major protein complex involved in mitochondrial calcium uptake is the
mitochondrial calcium uniporter (MCU) and, thereby, MCU inhibitors are
neuroprotective in different genetic models of chronic neurodegenerative
diseases

\textbf{Neuroinflammation}

In recent years, it has become clear that despite having many different
primary causes, all neurodegenerative diseases share a common hallmark:
neuroinflammation. Inflammation is the first line in host pathogen
defence and essential to the body's healing processes. However, chronic
or prolonged inflammation, like that observed in ageing and further
exacerbated in neurological diseases, is detrimental for tissue
homeostasis. Neuroinflammation can be triggered by CNS resident immune
and glial cells (e.g. microglia, astrocytes, oligodendrocyte lineage
cells), cells from the peripheral innate or adaptive immune system (e.g.
T cells, B cells, macrophages), meningeal inflammation, or
autoantibodies directed to the CNS.

\textbf{Neuroinflammation in ageing and neurodegeneration}

There is growing evidence showing that both, innate and adaptive immune
cells are present in the healthy CNS, where they have key roles in
maintaining homeostasis and immunosurveillance, being associated with
neurogenesis , learning and memory and synaptic pruning , among other
functions. However, this tightly regulated immune-CNS interaction is
distorted with ageing, and even more abruptly in neurodegenerative
disorders, leading to pathological neuroinflammation and subsequent
neurodegeneration . Even though present at low levels in the healthy
young CNS , an increase in adaptive immune cell infiltration, mainly
CD8\textsuperscript{+} T cells and to a lesser extent
CD4\textsuperscript{+} T cells, has been observed in the neurogenic
niches, the white matter, and the optic nerve with age. Enhanced T cell
infiltration alters CNS resident cell function increasing the expression
of interferon responsive genes in CNS stem and glial cells (neural stem
cells, microglia, and oligodendrocytes) and contributes to age-related
myelin degeneration, impaired neurogenesis, and axonal degeneration .
Increased CNS infiltration of peripheral immune cells with ageing, may
result from blood-brain-barrier alterations, increased permeability and
a decreased CNS perfusion and lymphatic drainage . Similar changes in T
cell infiltration have been also observed in a range of
neurodegenerative disorders not considered primary autoimmune disorders
such as AD, PD and ALS. Evidence has shown increased in T cell numbers,
especially CD8\textsuperscript{+} T cells, in the post-mortem CNS tissue
of AD, ALS and PD patients , and altered T cell levels or subsets in the
cerebrospinal fluid and peripheral blood AD, PD and ALS patients . The
role of adaptive immune cell-mediated inflammation in AD remains
controversial. Even if T cell depletion has rendered beneficial roles
reversing cognitive decline, increasing A\selectlanguage{greek}β \selectlanguage{english}clearance and promoting
neuronal survival , other studies have described detrimental roles for T
cells in AD pathology . In PD models on the other hand, mice lacking
mature lymphocytes show attenuated dopaminergic cell , while in an ALS
mouse model CD8\textsuperscript{+} T cells infiltrate in the CNS are
associated with motor neuron loss . Thus, the role of T cell-mediated
neuroinflammation in neurodegenerative diseases may be subset and
context specific, highlighting the complexity of the CNS-immune
crosstalk and the role of neuroinflammation in neurodegeneration.
Prolonged CNS immune infiltration together with the enhanced production
of pro-inflammatory cytokines (e.g: IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-6 or IL-1\selectlanguage{greek}β\selectlanguage{english})
described in both, ageing and neurodegenerative diseases, also
contribute to neurodegeneration indirectly, by perpetuating inflammation
through the priming of CNS glial cells. Single cell sequencing analysis
of CNS resident cells such as microglia, astrocytes and oligodendrocyte
lineage cells in different neurodegenerative diseases contexts and
ageing has unveiled disease-specific phenotypes characterized by the
expression of inflammatory and neurotoxic markers such as
\emph{Clec7a},\emph{C3, Lgals3, Trem2} in microglia , \emph{Serpina3n,
Lcn2, Ifitm3, Timp1, Chi3l}1 in astrocytes and Serpina3n, C4b, or Klk6
in oligodendrocyte lineage cells . Beyond this disease-specific
phenotype, an elevated expression of interferon responsive/stimulatory
genes (e.g:\emph{Irf1, Irf7, Irf8, Isg15, Ifit3}) has also been
described across glial cells in ageing and neuroinflammation .
Additionally, neuroinflammation enhances the expression of antigen
presenting genes (e.g: \emph{Cd74, B2m, Cd9, H2-K1, H2-D1}) as well as
immune cell chemoattractant cues such as \emph{Icam-1, Ccl2, Cxcl12 or
Ccl3} by all the main glial cells, which in turn further activate CNS
infiltrating T cells, contributing to a positive feedback loop that
perpetuates neuroinflammation and thus, neurodegeneration In addition to
immune cell infiltration and CNS resident cell-driven pro-inflammatory
reactions, another source of neuroinflammation is linked to the
accumulation of senescent cells with ageing and in CNS pathology.
Mounting evidence has demonstrated the accumulation of senescence
markers such as P16, P21, YH2A.X, lipofuscin, GATA4 and high-mobility
group box protein 1 in microglia, oligodendrocyte progenitor cells,
oligodendrocytes, astrocytes, and neurons with ageing and in pathology
such as AD . Senescent cells accumulate in aged tissues and contribute
to the pathogenesis of a range of neurodegenerative diseases, at least
in part, through their pro-inflammatory senescent associated secretory
phenotype (SASP) , which can propagate senescence to neighbouring cells
in a paracrine manner and contributes to immune cell recruitment to
eliminate senescent cells . Hence, neuroinflammation and senescent cells
establish an additional positive feedback loop exacerbating ageing and
disease pathogenesis: chronic inflammation like that observed in ageing
and neurodegenerative disorders enhances the appearance of senescent
cells, which in turn further contribute to neuroinflammation by the
secretion of pro-inflammatory molecules. Therefore, it is plausible that
either by eliminating senescent cells or by modulating SASP, we can
limit neuroinflammation and prevent neurodegeneration.

\textbf{Therapeutic approaches to counteract neuroinflammation}

Several drugs that limit immune cell infiltration in the CNS have been
developed in the context of MS, like Siponimod, Fingolimod or
Natalizumab However, whether these drugs are beneficial in other primary
neurodegenerative diseases such as AD or ALS is still under debate.
Natalizumab is a monoclonal antibody that blocks the extravasation of
immune cells in the CNS and has been proven successful in mouse models
of ALS such as SOD1\textsuperscript{G93A} and
TDP43\textsuperscript{A315T,} where it has diminished astrocyte and
microglia priming, increasing motor neuron number and survival .
Natalizumab has also shown beneficial effects in pre-clinical models of
AD such as APP/PS1 and 3xTg mice. In APP/PS1 mice Natalizumab reduced of
proinflammatory cytokines in the spleen, CD4 immunoreactivity and
general inflammation in the CNS . Similarly, in 3xTg-AD mice,
Natalizumab has improved memory and reduced microgliosis, A\selectlanguage{greek}β \selectlanguage{english}load and
tau hyperphosphorylation . Fingolimod on the other hand, is a structural
sphingosine analogue and sphingosine-1p-phosphate (S1P) receptor
modulator which blocks immune cell migration outside primary lymphoid
organs and thus, reduces T and B cell number in circulation. Fingolimod
has shown to promote survival and improve the phenotype in
SOD1\textsuperscript{G93A} mice and is well tolerated by patients with
ALS, although its efficacy in disease progression is yet to be evaluated
. In AD on the other hand, fingolimod has shown to ameliorate A\selectlanguage{greek}β
\selectlanguage{english}neurotoxicity in neuronal cultures while reducing A\selectlanguage{greek}β \selectlanguage{english}and neuronal loss
and astrocyte and microglial activation and improving memory and
learning deficits in 5xFAD mice . Furthermore, in PD mouse models such
as the MPTP mouse model and a model performed by intracerebral
injections of 6-hydroxydopamine, fingolimod attenuated
neuroinflammation, neuronal loss and motor deficits . Moreover, low-dose
of Fingolimod improved motor function and reduced brain atrophy, leading
to the extended survival of R6/2 mice, a mouse model of HD . Thus, even
though drugs that limit immune cell trafficking to the CNS appear to
have a beneficial effect and help preventing neurodegeneration in
different mouse models, further pre-clinical investigations followed up
by clinical trials are needed before clearly establishing their benefits
for patients in other neurodegenerative diseases beyond MS. An
alternative approach to limit not only immune cell mediated
inflammation, but also neuroinflammation mediated by CNS resident cells,
involves the use of other less specific anti-inflammatory drugs such as
minocycline or non-steroidal anti-inflammatory compounds. Minocycline is
a broad-spectrum antibiotic with important anti-inflammatory properties
and as such, it has been studied for several years now in mouse models
of neurodegeneration. Even if minocycline has been proven successful in
limiting neuroinflammation and in some cases neurodegeneration in mouse
models of AD , PD and ALS , its beneficial effect in subsequent clinical
trials has been less robust, with no clear neurocognitive improvement
observed in AD or HD, and disease worsening was detected in ALS trials .
Therefore, although most of the pre-clinical data using minocycline
reported positive results, its current negative outcomes or even the
symptom worsening observed in some clinical trials, questions its
effectiveness as a therapy for neurodegenerative diseases . Other
anti-inflammatory therapies considered for neurodegeneration include
non-steroidal anti-inflammatory drugs (NSAIDs), however, the potential
beneficial effects observed in some animal models, have not been
reproduced in clinical trials and thus their use as potential therapy
for neurodegeneration dropped . Considering the negative impact of
sustained inflammation, mammals have developed their own endogenous
anti-inflammatory break, which is mediated by regulatory T cells
(Tregs), a subset of immune cells with high immune suppressive capacity.
Tregs are known to be either depleted or functionally impaired in
multiple neurodegenerative disorders . Thus, systemic Treg expansion or
Treg adoptive transfer have been considered as a potential therapeutic
approach to tackle neuroinflammation and prevent neurodegeneration. Treg
expansion through peripheral interleukin-2/interleukin-2 monoclonal
antibody complexes or adoptive transfer upon \emph{ex vivo} activation
has rendered positive results in mouse models of ALS and AD, such as
SOD1\textsuperscript{G93A} mice , 5xFAD -Rag2Ko mice , 3-Tg-AD mice and
APP/PS1 mice . Tregs have been shown to protect motor neurons, supress
astrocytic and microglial immunoreactivity, reduce amyloid burden and
restore cognitive dysfunction. Moreover, an inverse correlation was
observed between Treg numbers and disease progression upon Treg
expansion in ALS patients, suggesting a neuroprotective effect also in
humans . Despite the ample evidence of a beneficial role for Treg having
in neurodegenerative diseases, systemic Treg expansion has not been
widely considered for clinical trials, as it can lead to systemic immune
suppression in patients of advanced aged and already vulnerable to
infections, limiting its therapeutic use. Recent investigations have
developed a gene delivery approach to locally expand Treg in the CNS by
overexpressing interleukin-2 in astrocytes, and thus, avoiding systemic
immune suppression. This adenoviral based gene delivery approach has
rendered positive results in mouse models of MS, stroke, and traumatic
brain injury . Even though still to be tested in primary
neurodegenerative disorders, such as ALS, PD or AD, this approach opens
novel therapeutic venue to harness Treg immunosuppressive capacity to
limit CNS neuroinflammation and neurodegeneration. Last, we review the
potential use of senolytics to limit neuroinflammation and prevent
neurodegeneration. The fact that mice genetically engineered to remove
p16\textsuperscript{INK4a+} senescent cells show a decrease in
age-related pathologies in several tissues, together with an extended
lifespan and health span , boosted the interest in the development of
senolytics, such as dasatinib, digoxin or quercetin, as potential
therapeutic approaches for neurodegeneration . Senescent oligodendrocyte
progenitor cells (OPC) have been found around A\selectlanguage{greek}β \selectlanguage{english}plaques in post-mortem
tissue of patients with mild-cognitive impairment or AD and APP/PS1
mice. The elimination of senescent OPCs by the administration of the
senolytic cocktail formed by dasatinib and quercetin, decreased
microglial activation, A\selectlanguage{greek}β \selectlanguage{english}load, and the concentration of inflammatory
cytokines IL-6, IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}and improved cognitive performance .
Additionally, elimination of senescent astrocyte and microglia via the
administration of senolytic AP20187 in MAPT\textsuperscript{P301S}PS19
tau-pathology mouse model prevented gliosis, hyperphosphorylation of tau
and neurodegeneration and preserved cognitive function . Similarly,
removal of senescent microglia via AP20187 or dasatinib and quercetin
administration prevented age-related cognitive decline and
neuroinflammation . Thus, senolytics prevent neurodegeneration in ageing
and pre-clinical models of AD, supporting their use in clinical trials
with older adults suffering from MCI or early-stage AD (NCT04685590.
SToMP-AD and NCT04785300, ALSENLITE) , However, their beneficial effect
in other neurodegenerative diseases such as ALS or PD is yet to be
investigated. Even though the pre-clinical results in AD and ageing look
promising, the use of senolytic approaches to eliminate senescent cells
should be considered cautiously, due to the lack of knowledge regarding
the role of senescent cells in neurodegeneration. One alternative to
avoid the potential negative effects of eliminating senescent cells is
to focus on developing therapies aiming at reducing or eliminating SASP
to limit neuroinflammation . Senomorphics or SASP inhibitors can limit
senescent cell SASP production by inhibiting NF-kB, JAK-STAT, mTOR or
mitochondrial complex I and IV related targets. However, since
senomorphics do not eliminate senescent cells, continuous treatment with
SASP inhibitor would be required to obtain long-lasting effects, which
could also increase off-target effects associated with the suppression
of cytokine secretion by other cells. Thus, a better understanding of
the role of SASP and senescent cells in the CNS diseases and ageing is
essential to successfully develop senotherapeutic interventions to limit
neuroinflammation and target neurodegeneration .

\textbf{Conclusions and future perspectives}

We have attempted to briefly review the enormous field of the biological
pathways that are affected during ageing and implicated in the
pathogenesis of neurodegenerative disorders. Even though a common
feature of neurodegenerative diseases is the abnormal deposition and
mis-localization of insoluble protein aggregates, different cellular
pathways contribute to neuronal loss. Furthermore, these pathways are
all affected by ageing, which represents the main common risk factor for
most neurodegenerative disorders. Cells in all regions of the CNS are
affected by ageing, as indicated by the decline of sensory, motor, and
cognitive functions with time . However, how cellular and molecular
changes that occur during normal ageing render neurons vulnerable to
degeneration has not yet been fully elucidated. Currently, most efforts
to treat neurodegenerative disorders focus on strategies that target the
insoluble aggregates of proteins specifically associated with each
neurodegenerative disorder. So far, most of the clinical trial results
have been disappointing because cognitive function is not restored even
when protein aggregates are removed. These results point towards the
importance of studying the cellular pathways that contribute to neuronal
dysfunction, in order to provide combined therapies to the patients.
Neuronal function requires an efficient network of pathways that are
strictly connected and interdependent. For instance, synaptic function
is affected by the inflammatory microenvironment generated by the ageing
glial cells. Furthermore, synaptic transmission requires energy and
perturbed mitochondrial function has been associated with ageing and
neurodegeneration as the quality control of the cellular components is
regulated by energy sensors. Additionally, the lysosomal-dependent
self-digestive process of damaged proteins and organelles, called
autophagy, is important to generate nutrients and energy to maintain
essential cellular activities. Defects in autophagy results in
intracellular protein accumulation, contributing to the formation of the
insoluble aggregates of protein specifically associated to
neurodegenerative disorders. Finally, gene expression translates
synaptic activity and alterations in metabolic function into changes in
gene expression that can profoundly modify neuronal structure and
function. Different therapeutic strategies have been developed to target
these cellular pathways and the drugs have been evaluated for the
treatment of different neurodegenerative disorders. However, the future
challenges in drug discovery for neurodegenerative disorders are (i) the
detection of the earliest events in the neurodegenerative cascade and
(ii) the identification of the pathways responsible for the specific
vulnerability of cellular populations in each neurodegenerative disease.
The understanding of these mechanism is critical to develop
disease-modifying therapies and to design tailored therapies that can be
administered to specific patients' populations.\textbf{ReferencesFigure
legendFigure. 1} Scheme of the biological pathways contributing to
neurodegeneration

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